Precise epigenomic editing with a SunTag-based modular epigenetic toolkit

Abstract

Epigenetic regulation is a crucial factor controlling gene expression. Here, we report our CRISPR/dCas9-based modular epigenetic toolkit that enables gene-specific modulation of epigenetic architecture. By modifying the SunTag framework of dCas9 tagged with five GCN4 moieties, each epigenetic writer is bound to scFv and target-specific sgRNA, and this system is able to modify multiple epigenetic marks in a target-specific manner. We successfully demonstrated that this system is efficient in modifying individual histone post-translational modifications. We display its utility as a tool to understand the contributions of specific histone marks on gene expression by screening a large promoter region and identifying differential outcomes with high base-pair resolution. This epigenetic toolkit can be easily altered with a large variety of epigenetic effectors and is a useful tool for researchers to use in understanding gene-specific epigenetic changes and their relation to gene expression.

Keywords: CRISPR; Epigenetics; epigenetic writers; epigenome editing; histone post-translational modifications.

Figure 1.

Toolkit of Epigenetic Editors. a Schematic of epigenetic modulator design based on SunTag framework. 5xGCN4 sequences bound to dCas9 recruit epigenetic effectors fused with anti-GCN4 scFv antibody fragments to the sites directed by sgRNA. b Epigenetic modulators used in this study and their effects on histone marks. c Epigenetic editors alter histone marks in a site-specific manner. Each graph is normalized to input and represents biological triplicates.

Figure 2.

Screening of single gene promoter. a Scale diagram of promoter region of SNCA gene showing two alternative non-coding Exon 1 and Exon 2 with a start codon (ATG) as well as locations of sgRNA used. b Histone mark enrichment from our HEK293 dCas9 cell line for the indicated marks. (Peak locations are scaled to diagram shown in a. Average PCR bin size = 275 ± 25 base pairs). c qPCR assay was carried out to determine the change in α-synuclein expression levels relative to empty scFv control (Set to 1) for JARID1A and EZH2 targeted to specified loci by sgRNA. (Locations are scaled to diagram shown in a). Each point is normalized to empty control and represents biological triplicates. d,e Histone mark changes after transfection with indicated sgRNA and d JARID1A-scFv or e EZH2-scFv. Black lines indicate location of the sgRNA targeting site on the chromosome. Average PCR bin size = 275 ± 25 base pairs. f qPCR assay shows the change in α-synuclein expression levels relative to empty scFv control for JARID1A and EZH2 targeted separately or together to a single sgRNA target site shows that combined targeting is more effective than either alone. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.