Some scientific breakthroughs are so transformative that there’s no containing their spread once they’ve seeded. By engineering cellular nuclear reactions with a biochemical twist, the most powerful deactivated Cas9 (dCas9) methyltransferase explodes from the lab of Tomasz Jurkowski at the University of Stuttgart. The free energy of this reaction forges not only a new tool but also unprecedented knowledge of DNA methylation’s mechanisms.
First author Peter Stepper and Senior Author Tomasz Jurkowski share, “We wanted to study the epigenetic and transcriptional consequences of targeted DNA methylation. Instead of using a simple fusion of dCas9 to the Dnmt3a catalytic domain (CD), we used a highly active chimeric fusion between the methyltransferase (MTase) domain and its natural activator Dnmt3L. This turned out to be an extremely efficient construct to deposit methylation (with up to 5-fold increased methylation efficacy) on targeted loci.
Here’s what went down when targeting CpG islands in the promoters of EpCAM, CXCR4, and TFRC in human cell lines (SKOV-3 and HEK293):
- Bisulfite sequencing revealed induced DNA methylation at targeted promoters and RT-qPCR detected decreased gene expression.
- A single sgRNA resulted in efficient and widespread DNA methylation at a targeted region; although, multiplexing several sgRNAs didn’t increase efficiency.
- dCas9-Dnmt3a-Dnmt3L produced 4 to 5 times more methylation than dCas9-Dnmt3a.
- The methylation profile produced displayed a distinct profile:
- 25 bp upstream from the PAM site displayed the strongest peak
- 40 bp downstream from the PAM exhibited another weaker peak
- The 20–30 bp around the dCas9 binding site is unmethylated, suggesting that dCas9 occupancy prevented methyltransferase action.
- The appearance of additional peaks ∼200 bp upstream and downstream of the PAM site may be due to nucleosome positioning.
Mechanisms of Methylation: DNMT Fibers
Not satisfied with just engineering the most potent designer DNA methyltransferase, the team then went on to provide novel insight into the basic mechanisms of methylation establishment and spreading.
Stepper and Jurkowski continue, “Once we did the targeting experiments we noticed that there is extensive spreading of methylation which reached close to 1000 bp away from the Cas9 binding site. Then, as biochemists, we wondered what could be the mechanism behind this and it had to be something different than simple flexibility of the linker between dCas9 and the MTase. We thought that this actually could be achieved via looping of the DNA to reach distant regions in the locus or (and maybe “and”) multimerization of the Dnmt3a CD, a feature that we knew about from biochemical experiments.
“Luckily, we also knew which mutation to introduce in the Dnmt3a domain to prevent this multimerization and we checked that in the targeting setup in the cells. We were very happy to see that the mutant methylated the CpG sites directly adjacent to the target sites and yet left the distant sites unmodified, therefore linking the multimerization to spreading. It is really amazing to see what a single mutation of a non-catalytic residue can change in the function of the enzyme. It would also explain how DNA methylation of complete CpG islands can be achieved by recruiting the MTase by e.g. transcription factors to a single place within the CGI with the MTase serving as a kind-of nucleation point from which the methylation could spread.”
“This also showed us that the targeted epigenetic editing is a fantastic approach to study variants or mutants of epigenetic writers in the cellular environment, allowing us to study their effects on a defined locus with minimal pleiotropic effects (compared to simple overexpression), which I don’t think anybody else exploited so far.”
Epigenetic Editing Outlook
The talented duo concludes, “Regarding the future of epigenetic editing, efficient systems to target multiple genomic locations with different editing activities in the same cell are required to gain control over cell differentiation or to study epigenetic wiring in a more comprehensive manner. We expect that the next wave of discoveries will be related to the wiring of the epigenetic network with targeted studies, approaching the stability of the epigenetic marks and finding combinations of epigenetic effectors that lead to a stable epigenetic switch or looking at the crosstalk between epigenetic marks (like DNA methylation and histone modifications). Thereby, one could think of CRISPR-based epigenetic editors as a picklock to crack the epigenetic code.”
Go learn how to seed a methylation chain reaction on your favorite gene at Nucleic Acids Research, November 2016