Fig. 4.

The expression of GBP4 in PDAC cells was downregulated by targeted methylation with dCas9-SunTag-DNMAT3A-sgGBP4 system. A Schematic of dCas9-SunTag-DNMT3A1-sgGBP4. Deactivated Cas9 (dCas9) was fused to SunTag epitopes and the single-chain variable fragment (scFv) was fused to green fluorescent protein (GFP) and DNMT3A1. Multiple copies of scFvDNMT3A1 can be guided by single guide RNA (sgRNA) to recognize specific loci and methylate regions of interest. B The recognition sites of sgRNAs used to guide the dCas9-SunTag-DNMT3A1 in the regulatory regions of human GBP4, with arrows (aligned to the magnified regions) indicating 20 bp binding sites of sgRNAs. Arrows point toward the PAM sequence. C, D The methylation status of CpG sites in the regulatory regions of GBP4 gene in Capan-1 cells and AsPC-1 cells were analyzed using bisulfite genomic sequencing after transfection using the dCas9-SunTag-DNMAT3A-sgGBP4 system, respectively. E The mRNA expression of GBP4 in Capan-1 cells and AsPC-1 cells were measured using quantitative real-time PCR after transfection using the dCas9-SunTag-DNMAT3A-sgGBP4 system, respectively. The data are shown as mean ± SD; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001