Figure 1
From: Targeted DNA methylation in human cells using engineered dCas9-methyltransferases
RNA-guided CpG methylation in E. coli. (a) Model of dCas9 and M.SssI bound to the same DNA at a gap of 11 base pairs between the PAM and the target CpG site. Model was built by superimposing the crystal structures of DNA-bound dCas9 (4UN3)37 and the M.SssI homolog M.HhaI (2HR1)55 on the same molecule of B-DNA. M.HhaI is bound to the trans strand relative to the PAM site. Red indicates the methylated cytosine, which is flipped out of the dsDNA. A hand-drawn black line represents the 15-amino acid linker between the C-terminus of dCas9 (orange) and the N-terminus of MC (green). The linker was not explicitly modeled. (b,c) Side views of the model with M.HhaI bound to the trans or cis strand at a gap length of 11 base pairs. (d) Protein constructs used in E. coli studies. (e) Two-plasmid system for expressing dCas9-MC/MN and sgRNA and assaying methylation in E. coli. pReporter contains two target sites for methylation in which the CpG site is imbedded within an FspI site. (f) DNA sequence near the two target CpG sites for methylation indicating the PAM site (bold) and its distance from the CpG site (the gap). Full sequence of sgRNAs can be found in Supplementary Table S1. (g) Frequency of methylation at site 1 and site 2 on pReporter caused by dCas9-MC/MN as assayed by high-throughput bisulfite sequencing. The sgRNA expressed is indicated as is the no methylation control. sgRNA1* indicates that sgRNA1 was expressed, but that the site 1 protospacer and PAM sequences were scrambled. Values are the mean and the error bars are the standard deviation of three independent cultures. (h) Frequency of methylation at all off-target cytosines in CpG sites in pReporter as assayed by high-throughput bisulfite sequencing. The pattern of methylation on the plasmids can be found in Supplementary Figure S2.
