Silencing of human <i>HTT</i> by targeted CRISPR/dCas9-mediated epigenetic editing

BackgroundGene silencing is widely recognized as a promising therapeutic approach for dominant monogenic disorders. Current silencing strategies, many of which are transient, utilize RNA interference. Gene silencing may also be achieved through directed epigenetic editing using a CRISPR/dCas9 effector fused to DNA methyltransferase 3A (dCas9-DNMT3A). We used this system to direct DNA methylation to <i>HTT</i>, the causal gene underlying the autosomal dominant neurodegenerative disorder Huntington's disease, to assess the translational potential of this strategy for treating a genetic neurological disease.ObjectiveTo characterize the regulatory effect of targeted dCas9-DNMT3A-mediated DNA methylation at <i>HTT</i>.MethodsWe exploited DNA methylation profiles of high and low <i>HTT</i>-expressing tissues and targeted hypomethylated regions of <i>HTT</i> associated with high levels of <i>HTT</i> expression.Results<i>De novo</i> DNA methylation of loci within defined upstream, promoter, intragenic and downstream regions of <i>HTT</i> resulted in robust, acute silencing of <i>HTT</i>. The best long-term silencing of <i>HTT</i>, which persisted up to 30 days, was observed when targeted DNA methylation was directed to the 5'UTR and promoter regions of <i>HTT</i>.Conclusions<i>HTT</i> gene silencing may be achieved via targeted <i>de novo</i> DNA methylation within hypomethylated regulatory regions at the <i>HTT</i> locus. DNA methylation editing may be an attractive therapeutic approach for Huntington disease due to its potential for long-term silencing and reversibility.