A modular dCas9-based recruitment platform for combinatorial epigenome editing

Targeted epigenome editing tools allow precise manipulation and investigation of genome modifications, however they often display high context dependency and variable efficacy between target genes and cell types. While systems that simultaneously recruit multiple distinct 'effector' chromatin regulators can improve efficacy, they generally lack control over effector composition and spatial organisation. To overcome this we have created a modular combinatorial epigenome editing platform, called SSSavi. This system is an interchangeable and reconfigurable docking platform fused to dCas9 that enables simultaneous recruitment of up to four different effectors, allowing precise control of effector composition and spatial ordering. We demonstrate the activity and specificity of the SSSavi system and compare it to existing multi-effector targeting systems, demonstrating its efficacy. Furthermore, we demonstrate the importance of the effector recruitment spatial ordering for effective transcriptional regulation. Together, the SSSavi system enables exploration of combinatorial effector co-recruitment to enhance manipulation of chromatin contexts previously resistant to targeted editing. Overall design: Chromatin immunoprecipitation sequencing (ChIP-seq) for histone modifications H3K4me3, H3K9me3 and H3K27me3 in HEK293T cells stably expressing dCas9-SSSavi and 6x sgRNA, and transfected with either DNMT3A+KRAB+EZH2 (D3A-K-E) or aGCN4-mCherry (non-catalytic control) for 72h, followed by flow cytometric enrichment of 200,000 cells positive for GFP (containing SSSavi components). WGBS was performed on HSB6G cells transfected with either D3A-K-E or ??GCN4-mCherry for 72h, followed by flow cytometric enrichment of 100,000 cells positive for GFP (containing SSSavi components). Genomic DNA was extracted and processed as detailed in the protocols section.