dCas9-based epigenome editing suggests acquisition of histone methylation is not sufficient for target gene repression

Abstract

Distinct epigenomic profiles of histone marks have been associated with gene expression, but questions regarding the causal relationship remain. Here we investigated the activity of a broad collection of genomically targeted epigenetic regulators that could write epigenetic marks associated with a repressed chromatin state (G9A, SUV39H1, Krüppel-associated box (KRAB), DNMT3A as well as the first targetable versions of Ezh2 and Friend of GATA-1 (FOG1)). dCas9 fusions produced target gene repression over a range of 0- to 10-fold that varied by locus and cell type. dCpf1 fusions were unable to repress gene expression. The most persistent gene repression required the action of several effector domains; however, KRAB-dCas9 did not contribute to persistence in contrast to previous reports. A ‘direct tethering’ strategy attaching the Ezh2 methyltransferase enzyme to dCas9, as well as a ‘recruitment’ strategy attaching the N-terminal 45 residues of FOG1 to dCas9 to recruit the endogenous nucleosome remodeling and deacetylase complex, were both successful in targeted deposition of H3K27me3. Surprisingly, however, repression was not correlated with deposition of either H3K9me3 or H3K27me3. Our results suggest that so-called repressive histone modifications are not sufficient for gene repression. The easily programmable dCas9 toolkit allowed precise control of epigenetic information and dissection of the relationship between the epigenome and gene regulation.

Guidelines for Optimized Gene Knockout Using CRISPR/Cas9

“…C15200216). (B) HEK293T cells were transfected with dCas9 plasmid (Addgene #100091) [43]. dCas9 localization was analyzed by immunostaining using anti-Cas9 mouse monoclonal antibody (Diagenode Cat No.…”

Clinical epigenetics: a primer for the practitioner

“…50 Targeting the proteins mutated in these syndromes or understanding the specific epigenetic abnormalities causing these syndromes should help us develop specific treatments for these disorders. Given the complexity and rarity of these conditions, it may be years until they are properly identified and understood.…”

DNA methylation in epigenetic inheritance of metabolic diseases through the male germ line

“…If introduced in a cellular context together with single guide RNA (sgRNA), this system can be used to direct transcriptional repression or activation to specific loci through the specificity of the sgRNA sequence (Konermann et al 2015, Shalem et al 2015, O'Geen et al 2017. The CRISPR-Cas9 system has also been modified to direct single loci-specific de novo DNA methylation by fusing dCas9 with DNMT3A (Liu et al 2016, McDonald et al 2016, Stepper et al 2016, Vojta et al 2016, O'Geen et al 2017 or DNA demethylation by fusing dCas9 with TET1 (Choudhury et al 2016, Liu et al 2016. Despite the continuing lack of an exhaustive clarification of eventual off-target effects, CRISPR-dCas9 systems could allow a future functional description of the importance of the methylation status of individual or clusters of CpG sites for epigenetic inheritance of metabolic diseases.…”