Figure 1. Structure of the human p16INK4a gene in HCT116.

(a) The Gx4 allele is not transcribed because the CpG island (including the promoter region, first exon, and first intron) is CpG-methylated. In the Gx5 allele, a frameshift mutation caused by insertion of a single guanine (G, shown in red) in the coding region of the first exon prevents production of the functional protein. The Gx4 and Gx5 sequences are shown in uppercase. (b) The CpG island of the p16INK4a gene. (Upper) Schematic diagram of the CpG island around the first exon of p16INK4a. Four alternatively spliced mRNAs are transcribed from the CDKN2A locus, one of which is p16INK4a. The CpG island is shown in green. (Lower) DNA sequence of the CpG island in the Gx4 allele. An additional guanine (G) is inserted into the G stretch (shown in uppercase) of the Gx5 allele. The upper image and DNA sequence were generated using the UCSC Genome Browser (https://genome.ucsc.edu/). CpG sites are underlined. (c) Primer positions for bisulfite sequencing. (d) Bisulfite sequencing of genomic DNA extracted from HCT116. The target sites for sgRNA_lef5, sgRNA_mid2, and sgRNA_rig3 are shown in purple, red, and light blue, respectively (b–d).