Gene Repression Via CRISPR-Assisted Modification of Specific Chromatin Marks

The CRISPR-CAS9 system allows for efficient and specific nuclease targeting, simply by programming target site recognition with a short guide RNA. Fusions of its deactivated variant (dCas9) are being used widely to activate and repress gene expression in a highly specific and robust manner when targeted to gene promoters and enhancers. For example, dCas9 fused to either the VP64 activation domain, the KRAB repression domain, or the P300 acetyltransferase domain have been shown to alter the modification of chromatin marks H3K4, H3K9, and H3K27 in a context-dependent manner. In this current study, we developed fusions of dCas9 to other various chromatin modifiers (SETDB2, SIRT6, EHMT2) and demonstrated their transcriptional repression of the genes MeCP2, FMR1 and SOX2. Additionally, we’ve characterized the chromatin changes that those fusions deposit. In particular, the Sirt6 fusion removes acetyl marks from H3K9 and H4K16 residues, while the SETDB2 fusion catalyzes targeted tri-methylation of the H3K9 residue, but does not affect H3K27 marks. The latter contrasts with the more commonly used KRAB-based repressor, which recruits a repressor complex that leads to accumulation of a variety of repressive histone marks. Ongoing studies include exploring the combinatorial effect of depositing multiple specific chromatin marks at the same locus and how this translates to changes in gene expression. These fusions expand the epigenome-engineering toolkit and will facilitate more specific manipulation of chromatin and understanding its role in gene regulation, which ultimately may also enable more deterministic programming of gene expression.