Figure 5: Evidence for HR-mediated repair of telomeric DSBs.

(a) Cells were prepared as described in Fig. 4b. Telomeres were visualized using FISH. A representative image shows large foci, indicating clustering of telomeres in cells with telomeric DSBs. Scrambled sgRNA was used as a control. Scale bar, 10 μm (b) Quantification of a, showing the average number of telomere foci in CRISPR-Cas9-targeted cells. Values are the average ±s.d. of three independent experiments (n=100). ^***P<0.001. (c) Cells were prepared as in Fig. 4a. DNA was prepared and analysed by 2D agarose gel electrophoresis, followed by hybridization with a G-rich telomere strand-specific probe under native or denatured conditions. ExoI treatment of DNA prior to 2D gel electrophoresis eliminated the signal from ss-C-rich DNA. (d) Quantification of the C-overhang. Values are the average ±s.d. of three independent experiments. *P<0.05. (e) Quantification of the C-overhang sensitive to ExoI digestion. Values are the average ±s.d. of three independent experiments. *P<0.05. (f) Detection of T-SCE by CO-FISH (see methods). Yellow dots indicate the presence of T-SCE. Cells were transfected with telomeric sequence (Telo) or scrambled sgRNA (Scr) as indicated. Scale bar, 20 μm. (g) Summary of CO-FISH results. The Student’s t-test was used to determine the statistical significance.