485. Targeted Epigenome Editing by CRISPR/Cas9-Based Repressors for Silencing of Distal Regulatory Elements

The CRISPR/Cas9 genome engineering platform has emerged as a powerful tool for gene regulation. Catalytically inactivated, “dead” Cas9 (dCas9) can be fused to a variety of transcriptional modulators to activate or silence gene expression. The KRAB domain, a common heterochromatin-forming motif in naturally occurring zinc finger transcription factors, has been genetically linked to dCas9 to create an RNA-guided synthetic repressor. KRAB-based synthetic repressors can effectively silence the expression of single genes and have been used to repress oncogenes, inhibit viral replication, and treat dominant negative diseases. However, the specificity of silencing endogenous genes and the distance of heterochromatin formation catalyzed by dCas9-KRAB repressors have not been studied. Understanding the precision of dCas9-KRAB repressors is critical to their further therapeutic application. As a model system to investigate the epigenetic consequences of KRAB-based silencing, dCas9-KRAB was targeted to the HS2 enhancer of the globin locus in the K562 human erythroid leukemia cell line. Enhancers are regulatory elements characterized by open chromatin that can activate distal genes targets. Genetic variation in enhancer sequences has been linked to many disease states, including cancer and cardiovascular disease. The HS2 enhancer is a potent activator of the developmentally regulated globin genes, which are involved in sickle cell anemia and beta thalassemia. We demonstrated that dCas9-KRAB targeted to HS2 by a single guide RNA (sgRNA) can disrupt the expression of multiple downstream globin genes. These findings expand the capabilities of synthetic repressors by showing that dCas9-KRAB targeted to a distal regulatory element can silence expression of multiple genes located 10 to 50 kb away. Genome-wide analysis of DNA-binding and mRNA expression by ChIP-seq and RNA-seq, respectively, established that RNA-guided synthetic repressors were highly specific for targeting and silencing the globin genes. To further investigate the utility of dCas9-KRAB repressors as a targeted epigenetic modifier, we performed ChIP-seq for tri-methylation of H3K9, a mark of heterochromatin, and DNase I hypersensitivity-sequencing (DNase-seq), a genome-wide measure of chromatin accessibility. dCas9-KRAB repressors induced H3K9 methylation at the HS2 enhancer. Off-target H3K9 methylation events were minimal and generally depended on the sgRNA. Reduced DNase I hypersensitivity was also observed at the enhancer region and its promoter targets, indicating that dCas9-KRAB repressors can modulate the chromatin structure of active enhancers to affect the activity of downstream target genes. These studies establish that dCas9-KRAB repressors can interfere with distal enhancer activity by highly specific remodeling of the epigenetic state of targeted loci. Enhancers play critical roles in differentiation, development, and disease, and understanding the epigenetic implications of targeting engineered repressors to distal regulatory elements will inform further use of genome engineering technologies for therapeutic applications.