Figure 4: Repair of CRISPR-Cas9-induced telomeric DSBs in 293T cells.

(a) DSBs were induced at telomeres by expression of telomeric sgRNA (Telo) in CRISPR-Cas9-transfected 293T cells. Scrambled sgRNA (Scr) was used as a control. DNA was isolated and analysed by CFGE as in Fig. 1c. (b) Immunofluorescence and FISH analysis was performed 24 h after CRISPR-Cas9 transfection of 293T cells. Normal 293T cells and cells exposed to scrambled sgRNA (Scr) were used as controls. Scale bar, 10 μm.(c) Quantification of b. The percentage of cells with one or more telomeric 53BP1 foci per cell was calculated. Values are the average ±s.d. of three independent experiments (n≥100). ^***P<0.001. The Student’s t-test was used to determine the statistical significance. (d) Telomere signal at chromosome termini determined using FISH. The percentage of chromosomes with one or more telomere-free ends was calculated, based on n=1,722 or 2,922 total chromosomes for Scr or Telo-sgRNA samples, respectively. Scale bar, 20 μm. (e) Cells treated as in b were analysed for SA-β-gal activity. Stress-induced 293T senescent cells were used as a positive staining control. Approximately 1,000 cells were counted and the percentage of senescent cells was calculated.