Is it a bird? Is it a plane? No, it’s a “super” new study that combines the strength, ability, and ingenuity of epigenetic editing to shut down our nemesis – the disease-associated dominant mutation – while leaving the innocent bystander allele unharmed!
Epigenetic superheroes led by Albert Jeltsch (University of Stuttgart) aimed to battle their nemesis by modifying a chimeric dCas9–Dnmt3a–Dnmt3L methyltransferase platform for allele-specific epigenetic editing. By improving components and designing single-guide (sg)RNAs for target regions in hypomethylated promoter CpG islands bearing a heterozygous single nucleotide polymorphism (SNP; linked to disease), they report a universal super-specific, allele-specific epigenetic silencing platform that can shut down the expression of alleles affected by a disease-associated dominant mutation while leaving the wild-type allele unaltered.
Let’s hear more from Rajaram and colleagues on their “super” new strategy to ensure allele-specific epigenetic silencing:
- An improved DNMT3A/3L catalytic component reduces non-specific activity while connecting sgRNA/dCas9 and DNMT3A/3L using SunTag affords signal amplification (10 DNMT3A/3L recruited by every dCas9) and facilitates DNMT3A/3L dimerization to ensure optimal catalytic activity
- The placement of SNPs in the PAM (trinucleotide proto-spacer adjacent motif) or seed regions of sgRNAs supports the design of 24 sgRNAs targeting single alleles at 14 different gene loci
- The application of the improved allele-specific epigenetic silencing platform induces efficient allele-specific DNA methylation for the ISG15, MSH6, GPD1L, MRPL52, PDE8A, NARF, DAP3, and GSPT1 gene loci in embryonic kidney cells
- Average DNA methylation levels vary across the targets; however, most successful targets reach an average DNA methylation level above 50% (indicating high efficiency)
- Variation could derive from sgRNA properties or the inherent epigenetic status of each region
- sgRNAs with the discriminatory SNP in the PAM region prompt a higher success rate and better specificity
- Any increases in DNA methylation at off-target alleles remain similar to alterations observed in controls
- sgRNAs with poor performance contain multiple PAM sites and quadruplex binding
- The highest expression of platform components and DNA methylation levels occurs three days after transfection
- Selected cases display allele-specific DNA methylation for up to 11 days (providing evidence for autonomous propagation of allele-specific DNA methylation) and a nearly 4-fold change in allelic expression ratios
Overall, these epigenetic superheroes flexed their muscles (and brains) and demonstrated their ability to take down their nemesis with their description of an allele-specific epigenetic silencing platform that supports the DNA methylation of target loci with “super” specificity, efficiency, and stability. Overall, this platform may allow the development of therapeutic approaches for diseases caused by dominant mutations by supporting mutant allele silencing while maintaining the expression of the wild-type allele.
For more on this super-specific epigenetic silencing platform, see Epigenetics & Chromatin, October 2023.