Figure 4: Assessing gRNA dosage and dCas9-HDAC3 range of activity at the Mecp2 locus.
From: Locus-specific histone deacetylation using a synthetic CRISPR-Cas9-based HDAC
(a) The Mecp2 promoter locus is schematically depicted with the approximate locations of gRNA-3 and the ChIP-qPCR amplicon regions (in blue). (b) H3K27ac ChIP-qPCR enrichment (relative to dCas9-HDAC3R265P) at regions in the Mecp2 promoter depicted in Fig. 2c. One-way ANOVA with Dunnett post-test; * P<0.05; **P<0.01; n=4 biological replicates; error bars, s.e.m. (c) H3K27ac enrichment at a genomic locus encompassing a 4 kb region around the Mecp2 TSS in dCas9-HDAC3R265P clonal cells. Green arrow indicates the position of gRNA-3. (d) Genomic region centred on the Mecp2 TSS (0=TSS) depicting the subtraction of the normalized H3K27ac uniquely mapped reads (RPM) in each dCas9-HDAC3 clone (clone 1: n=3 biological replicates; clone 2: n=2 biological replicates) from the normalized H3K27ac reads (RPM=reads per million) in the dCas9-HDAC3R265P clone (n=3 biological replicates). (e) H3K27ac RPKM values for the dCas9-HDAC3R265P clone (R265P), dCas9-HDAC3 clone 1 (HDAC3 clone 1) and dCas9-HDAC3 clone 2 (HDAC3 clone 2) at 17 potential off-target sites for Mecp2 gRNA-3 in the genome. (f) Relative mRNA expression of Mecp2 determined by RT–PCR by the indicated dCas9-HDAC3 clones co-expressing Mecp2 gRNA-3 (relative to dCas9-HDAC3R265P also co-expressing Mecp2 gRNA-3) One-way ANOVA with Dunnett post-test; ****P<0.0001; n=7 biological replicates; error bars, s.e.m.
