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. 2026 Feb 5;54(4):gkag089. doi: 10.1093/nar/gkag089

Figure 1.

Figure 1.

Genome-editing of Halo-tagged DNMT1 and determining number of DNMT1 molecules per cell. (A) Schematic of CRISPR-editing for inserting sequences encoding the Halo-3XFLAG tag into the 3’ ends of DNMT1 endogenous loci in U2OS cells. Red arrows, primer pairs used for PCR verification. (B) PCR verification of CRISPR-edited U2OS cell lines. gDNA was extracted from individual clones following puromycin selection and cell sorting of JF549-Halo dye-labelled cells. Primers are F = forward; R = reverse. (C) Top, 3–8% Tris-acetate gel stained with JF646 showing the presence of DNMT1-Halo-FLAG in the E8 (homozygous positive) and F3 (heterozygous) cell lines with parental U2OS cells as a control. Wedge, 2x serial dilutions. Middle, immunoblot showing the levels of endogenous (marked with *) and DNMT1 Halo-FLAG. The 70 kDa band on the Ponceau stain of the same blot was used as a loading control. (D) Flow cytometry estimating the abundance of Halo-tagged DNMT1 in E8 (+/+) and F3 (+/-) cell lines relative to Halo-CTCF in different U2OS cells. Two biological replicates are shown for both JF646-labeled and unlabeled cells. (E) Relative abundance of DNMT1-Halo and Halo-CTCF, endogenous (untagged) DNMT1 in the indicated cell lines, also see Supplementary Tables 1-3.(F) Percent methylation of CpG dinucleotides in genomic DNA determined by nanopore sequencing.