a, Schematic of CRISPR–Cas9-based generation of CTCFY226A/F228A cells. The guide targets cleavage of exon 1 of the CTCF gene. The repair oligonucleotide renders the gene noncleavable by Cas9, and simultaneously introduces mutations in the codons that encode Y226 and F228. b, The CTCFY226A/F228A mutation was confirmed by Sanger sequencing, including a silent mutation at position 229. c, Western blot depicting Halo-tagged SCC1 in wild-type and CTCFY226A/F228A cells. The parental wild-type cells are included as a control. This experiment was performed once. d, Representative images of cells in G1 and G2, as indicated by their nuclear and cytoplasmic localization of DHB–iRFP, respectively. e, Chromatin-bound levels of CTCF and SMC1 analysed by western blot. Histone H4 is used as a control for the chromatin fraction. The CTCFY226A/F228A mutation does not evidently affect overall CTCF and cohesin levels on chromatin. WCE, whole-cell extract; CB, chromatin-bound fraction. This experiment was performed twice with similar results. f, Relative SCC1–Halo fluorescence intensity quantified in the unbleached area directly after photobleaching, as a proxy for the chromatin-bound fraction of SCC1. This nondiffusive fraction is not evidently affected by the CTCFY226A/F228A mutation. Individual cells of three independent experiments are plotted as dots and their mean is indicated (21 wild-type cells and 17 CTCFY226A/F228A cells were scored).