A modular dCas9-SunTag DNMT3A epigenome editing system overcomes pervasive off-target activity of direct fusion dCas9-DNMT3A constructs

Modular dC9Sun-D3A system outperforms dC9-D3A direct fusion. (A) Titration of αGCN4-D3A effector (D3A [human DNMT3A catalytic domain]). Fraction of mCG is shown in black bars; dotted lines and black arrows indicate region used to calculate mCG change. (B) Comparison of dC9-D3A high, dC9-D3A, dC9Sun-D3A, and dC9Sun-mCherry (CRISPRi control) at the UNC5C promoter (on-target) versus the BCL3 promoter (off-target) by targeted bsPCR-seq (average mCG/CG, n = 3 replicates; error bars, SD). (C) mCG deposition efficiency by dC9Sun-D3A at three different loci (CCDC85C, SHB, and UNC5C promoters) measured by targeted bsPCR-seq (average mCG/CG, n = 3 replicates; error bars, SD).