Skip to main content
Fig. 5 | Genome Biology

Fig. 5

From: A novel two-step genome editing strategy with CRISPR-Cas9 provides new insights into telomerase action and TERT gene expression

Fig. 5

Single base-pair modification at the endogenous TERT promoter. a Outline of the protocol to modify a single base pair at the endogenous TERT promoter. First, a double-strand break was generated at the endogenous TERT promoter next to the targeted base pair with the CRISPR-Cas9 system (red scissors). Cells that underwent HR with the DT containing the SV40-driven eGFP expression cassette and the single base-pair modification were screened by GFP signal and confirmed by PCR with primers a’, b, c’, and e’, the sequences of which are listed in Table S3 in Additional file 1. HA homologous arm, TSS transcription start site. Next, the eGFP expression cassette was excised by two double-strand breaks generated by the CRISPR-Cas9 system. Cells that underwent HR with the DT containing the modified TERT promoter sequence were screened based on the loss of the fluorescent signal and sequencing the PCR products generated with primers a’ and e’. b Sample sequencing data demonstrating the reversion of the C-124T mutation from the TERT promoter in SCaBER cells. Peak color: red, T; green, A; blue, C; black, G. c TERT mRNA levels in the modified SCaBER clones are plotted relative to that in the parental cells (1.0, dashed line), as was measured by RT-qPCR with GAPDH mRNA as an internal control. The black line in each group of data points indicates the median value. As a group, the three clones had significantly reduced mRNA levels relative to the parental cells (p = 0.04 by Wilcoxon rank-sum test). d Telomerase activity in the modified SCaBER clones was lower than that in the parental cells, as measured by the IP-direct extension assay (LC loading control). Signal of the extension products on the gel was normalized to the LC signal and GAPDH protein levels in the IP input, which were quantified by western blot. The quantitative result is shown in the bar graph (mean ± standard deviation, n = 3 biological replicates, *p < 0.05 by Student’s t-test). e Telomeric restriction fragment length analysis by Southern blot in the parental cells and modified clones. Cells were harvested at the indicated time points. The average telomere length in each sample is plotted in the lower panel. *λ DNA-HindIII digest markers. f Growth curves for the parental cells and modified clones

Back to article page