Power, accuracy, and stability are the three tenets of hit-and-run epigenetic editing that every aspiring black belt must learn to master. To aid our quest, masters from the dojos of Luke Gilbert (University of California, San Francisco) and Jonathan Weissman (Massachusetts Institute of Technology) have come forth with not only new mantra but also a new set of moves: “CRISPRon, CRISPRoff”. These powerful new epigenetic editing tools were expertly engineered to program genome-wide transcriptional memory, leaving their mark on gene expression long after they’re gone. Take a look at the specs:
- CRISPRoff is composed of the KRAB, Dnmt3A (D3A), and Dnmt3L (D3L) domains fused to catalytically inactive S. pyogenes dCas9 to simultaneously deposit H3K9me3 and DNA methylation
- The KRAB domain remains at the dCas9 C-terminus but they positioned D3A-D3L at the N-terminus to optimize D3A accessibility for CpG sites
- Importantly, the domains are connected with proteolysis-resistant linkers, which prevent untethering of D3A and D3L in order to resolve one of the main causes of off-target DNA methylation with common D3A CRISPR systems
- The synergy of all three epigenetic repressors can impressively silence any gene with high specificity regardless of CpG island content in 80-90% of cells
- CRISPRon is a multi-partite epigenetic editor composed of TET1-dCas9 that co-recruits transactivator domains
- TET1 is relocated to the dCas9 N-terminus with an 80 amino acid linker peptide that improves DNA demethylation compared to common dCas9-TET1 systems
- To speed up reactivation kinetics, TET1-dCas9 is combined with a previously reported modified sgRNA encoding two MS2 stem loop sequences that recruit transactivator domains (VP64, p65-AD, and Rta) fused to the MS2 coat protein
- Reversal of CRISPRoff silencing with CRISPRon leads to near complete demethylation and reactivation of the target in more than 70% of cells within 2 days post-transfection
Now, let’s see these masters in action. Based on what’s been reported with previous epigenetic editing tools, transient CRISPRoff causes virtually no off-target effects and transient CRISPRoff silencing or CRISPRon reactivation is stable for weeks…even months!
- Transient CRISPRoff has minimal off-target effects on DNA methylation and gene expression
- There’s a non-specific increase of less than 2% in global DNA methylation, but there’s no spreading to neighbouring genes of the target
- There are detectable changes in expression for 1-3 non-target transcripts per knockdown experiment, but it doesn’t appear to be related to off-target DNA methylation
- However, an increase in H3K9me3 is detectable in neighbouring genes at comparable levels to the target
- Transient CRISPRoff and CRISPRon induce long-term epigenetic editing
- Transient CRISPRoff silencing persists for 15 months (about 450 cell divisions)
- Only 1 clone out of 39 shows reactivation of the target
- Transient CRISPRoff silencing in iPSCs even carries on throughout neuron differentiation
- Reactivation with CRISPRon is maintained for 28 days post-transfection, with Rta or p65-Rta transactivator combinations being most effective in the long-term
- Transient CRISPRoff silencing persists for 15 months (about 450 cell divisions)
“What was thought before this work was that the 30 percent of genes that do not have CpG islands were not controlled by DNA methylation,” said Gilbert. “But our work clearly shows that you don’t require a CpG island to turn genes off by methylation. That, to me, was a major surprise.”
The future of personalized medicine is brighter than ever with the coming of CRISPRoff and CRISPRon. Their ability to stably edit the epigenome while only there in passing makes them promising prospects for epigenetic therapy. CRISPRoff also kicks the door wide open for fundamental researchers by knocking out the need for knockouts when studying gene function and expression.
Kick-start your epigenetic editing strategy with CRISPRon and CRISPRoff in Cell, April 2021.