CRISPR-Mediated ZFP281 Degradation in ESCs

The CRISPR/Cas9 system was used to engineer ESCs for protein degradation of ZFP281, as previously described.^{52 (link)} The 5’- and 3’-homology arms of Zfp281 for C-terminal insertion were PCR amplified from genomic DNA. The 5’- and 3’-homology arms and FKBP12^F36V-2xHA-mCherry fragment were assembled by Gibson Assembly 2x Master Mix (NEB, E2611S). The CRISPR gRNA was subcloned into the pSpCas9(BB)-2A-Puro (PX459) vector (gRNA sequence in Table S3). ESCs were transfected with the donor and CRISPR vectors using Lipofectamine 3000 (Invitrogen). After 2 days of puromycin selection, mCherry-positive cells were seeded on a 96-well plate with single-cell per well using the BD Influx Cell Sorter. Cells were expanded and genotyped by PCR. Two clones (#2 and #21) with a homozygous knock-in were further expanded and used for experiments. Zfp281^degron ESCs and cEpiSCs were treated with dTAG13 (500 nM in DMSO, Tocris, 6605) for degradation of ZFP281 protein.