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. 2022 Mar 3;50(10):e59. doi: 10.1093/nar/gkac095

Figure 4.

Figure 4.

Antispacer PNAs modulate activity of dCas9-fusion systems. (A) POU5F1 expression over time in K562-dCas9-VPR/POU5F1-sgRNA cells treated with 250 pmol of non-targeting BFP antispacer PNA (blue) and POU5F1 antispacer PNA (red). Relative mRNA was measured by RT-qPCR and presented as fold-change relative to non-transduced parental K562 line. Models were fit using a smoothing spline, λ = 0.5. Shaded regions represent 95% CI. (B) MYOD1 expression over time in K562-dCas9-p300/MYOD1-sgRNA cells treated with non-targeting BFP antispacer PNA (blue) and MYOD1 antispacer PNA (gray). Relative mRNA was measured by RT-qPCR and presented as fold-change relative to non-transduced parental K562 line. Models were fit using a smoothing spline, λ = 0.5. Shaded regions represent 95% CI. (C) H3K27ac percent occupancy (calculated by percent input method with 2% input) at selected timepoints relative to PNA nucleofection (250 pmol dose). Parent K562 (no dCas9-p300, yellow) and stable K562-dCas9-p300 lines prior to nucleofection are labelled. (A), points represent average measurements for three technical replicates from two independent experiments (n = 2), mean differences and P-values for each timepoint listed in Supplementary Table S1. (B) points represent average measurements for three technical replicates from three independent experiments (n = 3), mean differences and P-values for each timepoint listed in Supplementary Table S1. (C) Bars represent mean ± s.e.m. from average measurements for three technical replicates from n= 3 independent experiments, mean differences and P-values listed in Supplementary Table S1.