HCT116 cells were transfected with D3A-dCas9/D3L and either KRAB-dCas9 or Ezh2-dCas9 and three gRNAs targeting the HER2 promoter. Control cells were transfected with dCas9 and the same three gRNAs. For transient repression timepoints, cells were cross-linked 5 days after transfection and growth in puromycin-containing media. For persistent repression timepoints, cells were sorted as described above and dCas9 control cells were cross-linked 24 days after transfection. Cross-linking was carried out in 1% formaldehyde for 10 min at room temperature and was stopped with 0.125 M glycine. Cross-linked cells were lysed with ChIP lysis buffer (5 mM PIPES pH8, 85 mM KCl, 1% Igepal) with a protease inhibitor (PI) cocktail (Roche). Nuclei were collected by centrifugation at 2,000 rpm. for 5 min at 4 °C and lysed in nuclei lysis buffer (50 mM Tris pH8, 10 mM EDTA, 1% SDS) supplemented with PI cocktail. Chromatin was fragmented using the Bioruptor 2000 (Diagenode) and diluted with 5 vol RIPA buffer (50 mM Tris pH 7.6, 150 mM NaCl, 1 mM EDTA pH8, 1% Igepal, 0.25% Deoxycholic acid). ChIP enrichment was performed by incubation with 3 µg H3K9me3 antibody (Diagenode C15410056), 2 µg H3K27me3 antibody (MP07–449), 2 µg H3K27ac antibody (Active Motif #39133) or 2 µg normal rabbit IgG (Abcam ab46540) for 16 h at 4°C. Immune complexes were bound to 20 µl magnetic protein A/G beads (ThermoFisher) for 2 hours at 4°C. Beads were washed 2x with RIPA and 3x with ChIP wash buffer (100 mM Tris pH8, 500 mM LiCl, 1% Deoxycholic acid). The final wash was performed in ChIP wash buffer with 150 mM NaCl. Cross-links were then reversed by heating beads in 100 µl ChIP elution buffer (50 mM NaHCO3, 1% SDS) overnight at 65°C and DNA was purified using the QIAquick PCR Purification Kit (Qiagen). Each entire ChIP sample was used to prepare Illumina sequencing libraries using the KAPA Hyper Prep Kit (Roche) and NEXTflex DNA barcodes (BIOO Scientific). Illumina sequencing libraries were pooled and sequenced using the HiSeq 4000 platform (Illumina) at the UC Davis DNA Technologies Sequencing Core.