Fig. 1

Evaluation of Casilio-ME1-mediated gene activation and 5mC demethylation. a Schematic representation Casilio-ME1 components. PUFa-TET1(CD) effector (TET1 residues 1418–2136), dCas9, and sgRNA with 3′extension scaffold comprising five PUFa-binding site (PBSa) are shown. Amino (N) and carboxyl (C) termini of protein fusions are arbitrarily shown. b Column plot showing fold changes in MLH1 mRNA levels in cells transfected with Casilio-ME1 components comprising MLH1-sgRNAs or NT-sgRNA. Cells were collected three days after transfection and were not subjected to selection. Error bars represent mean ± S.E.M (n = 3), data form two independent experiments are shown. NS, not significant, p > 0.05, two-way ANOVA. c Upper panel: MLH1 promoter and associated CpG island. Regions B and C are depicted according to a report correlating MLH1-silencing to region C hypermethylation³³. CpGs (lollipops), transcription start site (TSS) (arrow), and the sgRNAs used (A to F) are shown. Coordinates are relative to annotated TSS. Lower panel: high throughput BSeq analysis of MLH1 amplicons obtained from cells analyzed in (b). CpG methylation frequency of MLH1 promoter regions (mean ± S.E.M; n = 2) is shown. Arrows indicate locations of CpG overlapping the MLH1 sgRNAs (A–F) target sequences or TSS (blue arrow) as shown. CpG coordinates represent positions of cytosines, in base pair, relative to annotated TSS. p < 0.0001, two-tail t-test and two-way ANOVA. d DNA demethylation technologies compared in panel (e). TET1 effectors are tethered to dCas9 nucleoprotein at targeted site via binding of PUFa to PBSa, MS2 coat protein to stem-loop RNA structures appended to sgRNA, or ScFv (single-chain fragment variable) antibody against short peptide (GCN4) appended in array to dCas9 carboxy-terminus. TALE mediates delivery of TET1 activity via binding to targeted sequence. In MS2 system mouse TET1(CD) is also C-terminally fused to dCas9. e Evaluation of Casilio-ME1 platform as compared with alternative 5mC demethylation systems. MLH1 mRNA relative levels (mean ± S.E.M.; n = 3) in cells transfected with Casilio-ME1, MS2, TALE or SunTag components. Four MLH1-sgRNAs or NT-sgRNA were used with dCas9-based delivery systems. Four TALE effectors each targeting the sequences targeted by the MLH1-sgRNAs A, B, D, or F were used. ***p < 0.05, one-way ANOVA