Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements

Abstract

Epigenome editing with the CRISPR (clustered, regularly interspaced, short palindromic repeats)-Cas9 platform is a promising technology for modulating gene expression to direct cell phenotype and to dissect the causal epigenetic mechanisms of gene regulation. Fusions of nuclease-inactive dCas9 to the Krüppel-associated box (KRAB) repressor (dCas9-KRAB) can silence target gene expression, but the genome-wide specificity and the extent of heterochromatin formation catalyzed by dCas9-KRAB are not known. We targeted dCas9-KRAB to the HS2 enhancer, a distal regulatory element that orchestrates the expression of multiple globin genes, and observed highly specific induction of H3K9 trimethylation (H3K9me3) at the enhancer and decreased chromatin accessibility of both the enhancer and its promoter targets. Targeted epigenetic modification of HS2 silenced the expression of multiple globin genes, with minimal off-target changes in global gene expression. These results demonstrate that repression mediated by dCas9-KRAB is sufficiently specific to disrupt the activity of individual enhancers via local modification of the epigenome.

Figure 1. Silencing of downstream globin genes by dCas9-KRAB transcription factors targeted to the distal HS2 enhancer

(a) A panel of 21 sgRNAs were designed to target dCas9-KRAB to the HS2 enhancer, a distal activator of globin genes. Cr2, Cr4, Cr7, and Cr10 sgRNAs were selected for further study and stably delivered to K562 cells using the lentiviral vector shown in (b). (c–e) Repression of the HS2 enhancer was assayed by qRT-PCR of (c) HBE1, (d) HBG1/2, and e) HBB genes and fold-changes were calculated relative to non-transduced K562 cells (mean ± s.e.m). Within each panel, groups that share the same letter (A – F) are not significantly different by multi-way ANOVA followed by Tukey’s post-hoc test, P < 0.05 (n=3 independent experiments).

Figure 2. Specificity of gene regulation by dCas9-KRAB repressors targeted to the HS2 enhancer

RNA-seq was performed for genome-wide analysis of HS2 sgRNA silencing specificity. (a–d) Differential expression analyses demonstrate specific silencing of globin genes when comparing dCas9-KRAB targeted by (a) Cr4 and (b) Cr10 versus dCas9-KRAB without sgRNA and when comparing dCas9-KRAB guided by c) Cr4 and d) Cr10 to no lentivirus control (No LV CTL) K562s. Red data points indicate FDR < 0.01 by differential expression analysis compared to dCas9-KRAB controls without sgRNA (n = 3 biological replicates). Points labeled in blue indicate other globin genes.

Figure 3. Genome-wide binding activity of dCas9 repressors targeted to the HS2 enhancer

(a) ChIP-seq tracks demonstrate highly specific binding of FLAG-tagged dCas9 and dCas9-KRAB to the HS2 enhancer (shaded region) of the globin locus (chr11: 5244651 – 5314450), compared to dCas9-KRAB without sgRNA. An ENCODE K562 DNase I hypersensitivity DNase-seq track is included to highlight the globin LCR. (b,c) Differential analyses of global binding activity include comparisons of dCas9-KRAB targeted by (b) Cr4 and (c) Cr10 versus dCas9-KRAB without sgRNA. Points labeled in red indicate FDR < 0.05 by differential DESeq analysis (n = 3 biological replicates).

Figure 4. Genome-wide H3K9me3 signal in K562 cells treated with dCas9-KRAB targeted to the HS2 enhancer

(a) ChIP-seq tracks show increased H3K9me3 signal at the HS2 enhancer (shaded area) and flanking DHS sites in the globin LCR (chr11: chr11:5241410 – 5317466). An ENCODE K562 DNase I hypersensitivity DNase-seq track is included to highlight the globin LCR. (b,c) Global analysis of H3K9me3 patterns was performed by ChIP-seq for (b) Cr4 or (c) Cr10, comparing dCas9-KRAB with sgRNA versus dCas9-KRAB without sgRNA. Points labeled red indicate FDR < 0.05 by differential expression analysis compared to dCas9-KRAB without sgRNA (n = 3 biological replicates). (d) Counts for the HS2 enhancer (chr11: 5301862–5302715) from MACS-based peak calls were normalized to total counts (mean ± s.e.m). * indicated significance (p<0.05) by Student’s t-test compared to dCas9-KRAB only control.

Figure 5. Changes in global chromatin landscape with dCas9-KRAB localized to the HS2 distal enhancer

(a) Genome browser tracks of DNase-seq alignments at the globin locus (chr11: 5244651 – 5314450) show reduced DHS peaks at the HS2 and HS3 enhancers, as well as HBG1 and HBG2 promoter regions, in conditions containing dCas9-KRAB with sgRNA compared to dCas9-KRAB without sgRNA. Red shading labels the HBG1 promoter, HBG2 promoter, HS2 enhancer and HS3 enhancer regions for dCas9-KRAB + Cr4/10, which demonstrated decreased chromatin accessibility when compared to dCas9-KRAB with no sgRNA or dCas9 + Cr4/10. (b,c) Normalized DNase-seq cut counts within 800 bp window surrounding the (b) HBG2 promoter and (c) HS2 enhancer are shown (mean ± s.e.m, n = 3 biological replicates. * indicates p <0.05 compared to the dCas9-KRAB only sample (Student’s t-test). (d,e) Differential genome-wide analysis of changes in chromatin accessibility induced by dCas9-KRAB targeted by (d) Cr4 and (e) Cr10 compared to dCas9-KRAB without sgRNA in K562 cells. (f,g) Volcano plots of significance (p-value) versus fold-change for differential DESeq expression analysis of dCas9-KRAB guided by (f) Cr4 or (g) Cr10 compared to dCas9-KRAB without sgRNAs. Points labeled red indicate FDR < 0.05 by DESeq analysis. Points labeled in blue indicate other regions in the globin promoters or globin LCR.