CRISPR-Cas9 Deletion of Meg3 DMR CTCF Site

The guide RNA (sgRNA) was designed using CRISPR Design tool (zlab.bio/guide-design-resources) and synthesized with BbsI sticky ends: Meg3 DMR CTCF site 2—GTTGCACATAGAGACCGCTAG. It was cloned into the pUC57-sgRNA expression vector [49 (link)] (a gift from Xingxu Huang; #51132, Addgene). The Cas9-VP12 vector [50 (link)] (a gift from Keith Joung; #72247, Addgene) was modified by adding T2A-GFP at the C-terminal end and electroporated with the sgRNA vector into JB1 and BJ1 hybrid ES cells using the Amaxa nucleofector procedure (Lonza). Twenty-four hours post-electroporation, GFP-positive cells were sorted by flow cytometry (FACS Aria, Becton Dickinson) and single cells were seeded onto 96-well plates. After 10–12 days of culture, individual colonies were picked and grown in 6-well plates. Genomic DNA was extracted, and the region around Meg3 DMR CTCF site 2 was amplified (primers in Additional file 1: Table S2), followed by confirmation of the deletion by DNA sequencing (Additional file 1: Figure S6a).