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. 2019 Sep 20;10(1):4296.
doi: 10.1038/s41467-019-12339-7.

Enhanced CRISPR-based DNA Demethylation by Casilio-ME-mediated RNA-guided Coupling of Methylcytosine Oxidation and DNA Repair Pathways

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Free PMC article

Enhanced CRISPR-based DNA Demethylation by Casilio-ME-mediated RNA-guided Coupling of Methylcytosine Oxidation and DNA Repair Pathways

Aziz Taghbalout et al. Nat Commun. .
Free PMC article

Abstract

Here we develop a methylation editing toolbox, Casilio-ME, that enables not only RNA-guided methylcytosine editing by targeting TET1 to genomic sites, but also by co-delivering TET1 and protein factors that couple methylcytosine oxidation to DNA repair activities, and/or promote TET1 to achieve enhanced activation of methylation-silenced genes. Delivery of TET1 activity by Casilio-ME1 robustly alters the CpG methylation landscape of promoter regions and activates methylation-silenced genes. We augment Casilio-ME1 to simultaneously deliver the TET1-catalytic domain and GADD45A (Casilio-ME2) or NEIL2 (Casilio-ME3) to streamline removal of oxidized cytosine intermediates to enhance activation of targeted genes. Using two-in-one effectors or modular effectors, Casilio-ME2 and Casilio-ME3 remarkably boost gene activation and methylcytosine demethylation of targeted loci. We expand the toolbox to enable a stable and expression-inducible system for broader application of the Casilio-ME platforms. This work establishes a platform for editing DNA methylation to enable research investigations interrogating DNA methylomes.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
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 hypermethylation. 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
Fig. 2
Fig. 2
Casilio-ME2 mediates dual delivery of TET1(CD) and GADD45A to targeted genomic sites. a Schematic representation of the indicated Casilio-ME and Casilio platforms showing effector modules of PUFa fusion proteins used to transfect cells analyzed in (b). TET1(CD) (black), GADD45A (blue), p65HSF1 (green), PUFa (light gray), amino (N), and carboxyl (C) termini of protein fusions and occupancy of PBSa are arbitrarily shown. b Plot showing MLH1 mRNA relative levels (mean fold change ± S.E.M.; n = 3) in cells transfected with components of Casilio-ME1, Casilio-ME2.1, Casilio-ME2.2, or Casilio/p65HSF1 in the presence of MLH1-sgRNAs or NT-sgRNA as indicated. Cells were collected 3 days after transfection. Drawing of promoter regions with the MLH1-sgRNAs used (A-F), CpGs (lollipops), and TSS (arrow) is shown above the plot. ***p < 0.0005, one-way ANOVA. c Schematic representation of the indicated Casilio-ME platforms showing effector modules of PUFa and PUFc fusion proteins used to transfect cells analyzed in panel (d). TET1(CD) (black), GADD45A (blue), PUFa (light gray), PUFc (orange), sgRNA containing both PBSa and PBSc, amino (N) and carboxyl (C) termini of protein fusions are arbitrarily shown. d MLH1 mRNA relative levels (mean fold change ± S.E.M.; n = 3) in cells transfected with components of Casilio-ME1, Casilio-ME2.3, or Casilio-ME2.4 in the presence of MLH1-sgRNAs or NT-sgRNA is shown. When indicated PUFa-TET1(CD), effector component of Casilio-ME2.3 and Casilio-ME2.4, was replaced by a catalytically dead PUFa-TET1(CD) effector containing TET1-inactivating mutations H1671Y, D1673A, or omitted. MLH1 promoter with the sgRNAs used (A-F), CpGs (lollipops), and TSS (arrow) is depicted above the plot. ***p < 0.0005, one-way ANOVA
Fig. 3
Fig. 3
Casilio-ME3 mediates dual delivery of TET1(CD) and NEIL2 to targeted genomic site. a Illustration of the indicated Casilio-ME platforms showing effector modules of PUFa fusion proteins used to transfect cells analyzed in panel (b). TET1(CD) (black), NEIL2 (blue), PUFa (light gray), occupancy of PBSa, amino (N) and carboxyl (C) termini of protein fusions are arbitrarily shown. b MLH1 mRNA relative levels (mean fold change ± S.E.M.; n = 3) in cells transfected with components of Casilio-ME1, Casilio-ME3.1, or Casilio-ME3.2 in the presence MLH1-sgRNAs or NT-sgRNA as indicated. Drawing of promoter regions with the MLH1-sgRNAs used (A-F), CpGs (lollipops), and TSS (arrow) is shown above the plot. ***p < 0.001, one-way ANOVA. c Schematic representation of the indicated Casilio-ME platforms showing effector modules of PUFa and PUFc protein fusions used to transfect cells analyzed in panel (d). TET1(CD) (black), NEIL2 (blue), PUFa (light gray), PUFc (orange), sgRNA containing both PBSa and PBSc, occupancy of PBSa and PBSc, amino (N) and carboxyl (C) termini of protein fusions are arbitrarily shown. d Plot showing MLH1 mRNA relative levels (mean fold change ± S.E.M.; n = 3) in cells transfected with components of Casilio-ME1, Casilio-ME3.3, or Casilio-ME3.4 in the presence of MLH1-sgRNAs. Drawing of promoter regions with the MLH1-sgRNAs used (A-F), CpGs (lollipops), and TSS (arrow) is shown above the plot. ***p < 0.005, one-way ANOVA
Fig. 4
Fig. 4
Efficiency of 5mC demethylation induced by Casilio-ME platforms and dCas9-TET1 system. MLH1 promoter was targeted by using components of the indicated methylation editing system in the presence of MLH1-sgRNAs. Cells were collected 3 days after transfection and corresponding genomic DNA was subjected to high throughput amplicon BSeq and oxBSeq. a 5mC conversion to cytosine by TET1 and BER pathways is depicted above panels. Frequencies (mean ± S.E.M.; n = 2) of 5mC (upper panel), 5hmC (middle panel) and bisulfite converted CpG (C, 5fC, and 5caC) (lower panel) plotted against CpG positions within MLH1 promoter region are shown. The obtained levels of (5mC + 5hmC) and 5mC from two experiments were concordant. Arrows indicate locations of CpG overlapping one of the six sgRNAs target sequences. Statistical significance of differences in methylation patterns were tested. p < 0.0001, two-way ANOVA. b Box plot of frequencies of different CpG variants measured by BSeq and oxBSeq across the MLH1 promoter regions in cells transfected with the indicated 5mC demethylation system is shown. For each box plot the thick line inside the box represents the median value and the surrounding bottom and top lines represent the 25th and 75th percentiles. The whiskers represent min and max values, the x represents the mean value. **p < 0.01, and ***p < 0.0001, two-way ANOVA. c same as b but focusing on CpG 86–209 of the MLH1 promoter proximal-intron1 region. NS, not significant p > 0.05, and ***p < 0.01, two-way ANOVA

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