Protocol for Allele-Specific Epigenome Editing Using CRISPR/dCas9.

ABSTRACT

The discovery and adaptation of CRISPR/Cas systema for epigenome editing has allowed for a straightforward design of targeting modules that can direct epigenome editors to virtually any genomic site. This advancement in DNA-targeting technology brings allele-specific epigenome editing into reach, a "super-specific" variation of epigenome editing whose goal is an alteration of chromatin marks at only one selected allele of the genomic target locus. This technology could be useful for the treatment of diseases caused by a mutant allele with a dominant effect, because allele-specific epigenome editing allows the specific silencing of the mutated allele leaving the healthy counterpart expressed. Moreover, it may allow the direct correction of aberrant imprints in imprinting disorders where editing of DNA methylation is required exclusively in a single allele. Here, we describe a basic protocol for the design and application of allele-specific epigenome editing systems using allele-specific DNA methylation at the NARF gene in HEK293 cells as an example. An sgRNA/dCas9 unit is used for allele-specific binding to the target locus containing a SNP in the seed region of the sgRNA or the PAM region. The dCas9 protein is connected to a SunTag allowing to recruit up to 10 DNMT3A/3L units fused to a single-chain Fv fragment, which specifically binds to the SunTag peptide sequence. The plasmids expressing dCas9-10x SunTag, scFv-DNMT3A/3L, and sgRNA, each of them co-expressing a fluorophore, are introduced into cells by co-transfection. Cells containing all three plasmids are enriched by FACS, cultivated, and later the genomic DNA and RNA can be retrieved for DNA methylation and gene expression analysis.