Figure 5
From: A CRISPR-based approach for targeted DNA demethylation
Generality of gene targeted demethylation of the dCas9-based system. (a) Seven sgRNAs were designed to target the promoter region (−800 to −300-bp upstream of the transcription start site) of human MAGEB2 gene. (b) Quantitative real-time PCR (qRT-PCR) was carried out to assess the abundances of MAGEB2 mRNA in indicated groups. (c) Changes in the mRNA transcription of MAGEB2 gene were evaluated upon co-transfection of sgRNA (M1) with combinations of the indicated chimeric Tet1-CD or their respective mutants in HeLa cells. *P<0.05, compared with -sgRNA group by unpaired t-test. (d) The methylation states of DNA in the promoter region of MAGEB2 gene in each group were examined using bisulfite-sequencing approach and presented as above, with the detailed information listed in Supplementary Table S3. The sgRNAs recognizing their respective target sites were shown in blue-pink color (as in a). (e) Fourteen sgRNAs were designed to target sequences within the promoter region (−2 800-bp upstream of the transcription start site) of human MMP2 gene. Two transcript variants with different TSSs were shown here. (f) Activation of MMP2 mRNA transcription by sgMMP2-guided dCas9 demethylation system in HEK-293FT cells. The red dash line was used for easy comparison of the activation of mRNA transcription between the blank group and those of different groups. Data were shown after normalization to the controls (blank group) (means±s.e.m., n=3).
