Some of the best superheroes are born out of a mutational event. It’s how Peter Parker became Spiderman, how Bruce Banner turned into the Incredible Hulk, and where the X-Men derive their powers. It just goes to show that mutations aren’t always a bad thing — a point exemplified by the new superhero team in town: mutant DNMT3A alongside the SunTag system. Assembled together, they have the superpower of highly efficient epigenetic editing with reduced off-target effects.
Deep in their secret lair, a team led by Albert Jeltsch and Pavel Bashtrykov (Stuttgart University, Germany) set out to improve upon the current epigenetic editing systems that use targeted methylation to modify chromatin and silence genes. Just like with other epigenetic editing systems, they employed catalytically dead Cas9 (dCas9) and a guide RNA to direct a DNA methyltransferase to a target gene. Then they modified the system in three major ways. First, rather than just using the standard DNMT3A DNA methyltransferase, they used a DNMT3A/DNMT3L fusion that has a higher methyltransferase activity. Second, they combined this feature with the 10XSunTag system: dCas9 is fused to a SunTag, which acts as a scaffold for the recruitment of up to ten tagged DNMT3A/DNMT3L subunits. Finally, they also set out to further engineer the system to reduce off-target effects.
Using bisulfite sequencing, they measured methylation at the guide RNA-targeted ISG15 locus, as well as at an off-target site (VEGFA).
Here’s what they found:
- The SunTag system methylates the ISG15 locus efficiently (84% of CpG sites) but also methylates the off-target VEGFA locus (53% of sites)
- The off-target methylation is an effect of expressing DNMT3A/DNMT3L; the freely diffusing subunits of DNMT3A/DNMT3L can bind DNA independently of dCas9 and SunTag
- The crystal structure of DNMT3A bound to DNA revealed four critical residues that, when mutated, decrease the affinity of DNMT3A for DNA
- All four mutations decrease the off-target activity of DNMT3A/DNMT3L but also cause a decrease in the on-target methylation efficiency
- Combing mutant DNMT3A/DNMT3L with the SunTag system minimizes the off-target effects while retaining the most on-target activity
- The most promising mutation, R887E, retained 76% of the wild-type activity at ISG15 and had an 88% reduction in off-target VEGFA methylation
- In a genome-wide screen (MBD-seq), the R887E mutant has a 7.8 fold reduction in off-target methylation compared to the wild-type
By incorporating a mutation into DNMT3A, they created a more precise mechanism for programmable gene methylation. Although the addition of the SunTag system helps overcome some loss of activity by recruiting more effectors, more is not necessarily better. A longer SunTag that can recruit 24 effectors actually decreased the methylation efficiency.
So is this more precise system the hero we need in the epigenetic editing world? Will they be invited to join the Justice League? Read the full article and decide for yourself in the International Journal of Molecular Science, January 2020.