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Vector construction for targeted demethylation

TH Takuro Horii
SM Sumiyo Morita
SH Shinjiro Hino
MK Mika Kimura
YH Yuko Hino
HK Hiroshi Kogo
MN Mitsuyoshi Nakao
IH Izuho Hatada
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We have previously reported the dCas9 expression vector fused with five copies of GCN4 (pCAG-dCas9-5xPlat2AflD; Addgene plasmid 82560) and the expression vector for anti-GCN4 peptide antibody (scFv)-superfolder green fluorescent protein (sfGFP)-TET1CD fusion protein (pCAG-scFvGCN4sfGFPTET1CD; Addgene plasmid 82561) [31]. The gRNA vectors for H19-DMR and H19-promoter were generated by inserting the target sequences into gRNA cloning vector (Addgene plasmid 41824). Cloning was performed by linearization of an AflII site and Gibson assembly-mediated incorporation of the gRNA insert fragment. The gRNA target sequences are described (Additional file 1: Table S4 and S5). By contrast, two all-in-one vectors for H19-DMR (pPlatTET-gRNA2-H19DMRx9) and H19-promoter (pPlatTET-gRNA2-H19P) are based on pPlatTET-gRNA2 (Addgene plasmid 82,559). Detailed plasmid maps are shown (Additional file 2: Fig. S10–S12). Each all-in-one vector was linearized by ApaLI before use.

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