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. 2019 Aug 14;47(18):9637–9657. doi: 10.1093/nar/gkz709

Figure 5.

Figure 5.

Simultaneous epigenetic manipulation of the IL6ST and MGAT3 loci using DNMT3A-dSpCas9 and TET1-dSaCas9 in HEK293 cell line. (A) Fifteen CpG sites within CpG island of the IL6ST promoter were targeted with DNMT3A-dSpCas9, guided by the multi-guide system including four gRNAs, while 14 CpG sites located in CpG island 1 of the MGAT3 promoter were targeted with TET1-dSaCas9, guided by the multi-guide system consisting of six gRNAs. (B) Doubly transfected cells with the two dCas9 fusions showed concurrent increase and decrease of methylation level at the targeted CpG sites: 3–29% methylation change for IL6ST, and 19–72% methylation change for MGAT3, depending on the CpG site. (C) While change of methylation level in IL6ST promoter was followed with 0.583-fold change in transcript level (P = 0.0002 to inactive DNMT3A), the hypomethylation of 14 CpG sites in CpG island 1 of MGAT3 induced no increase of gene transcription. The human IL6ST locus (A) and MGAT3 locus (B) are shown with positions of the pyrosequencing assays (A1, A2) indicated by yellow rectangles. Magnified insets show individual CpG sites (white circles) targeted by dCas9 fusions and subsequently analyzed for methylation level. Arrows, aligned to the magnified regions, indicate 20 bp binding sites of gRNAs used to guide the dCas9-fusions. Arrows point toward the PAM sequence. Annotated promoter is represented by shaded red triangles; TSS, transcription start site (kinked arrow); A, dCas9 fusions with active catalytic domain; I, dCas9 fusions with inactive catalytic domain; NT, active dCas9 fusions with non-targeting gRNA. *** P < 0.001.