Fig. 1. Telomerase adds telomeric repeats to Cas9-induced DSBs.

(A) Schematic of pLenti-sgTS-TaqMan-TS. U6, human U6 promoter; sgLuc or sgTS, singleguide RNA (sgRNA) cassette; TS, PCR cassette containing the truncation sequence (TS); CMV, cytomegalovirus promoter; bsd, blasticidin S deaminase gene. The inset shows the sequence of TS PCR cassette, with the primers, TaqMan binding site, the patient-derived TS sequence, the Cas9 cleavage site, the sgRNA binding site, and the PAM highlighted. (B) Ethidium bromide (EthBr)–stained agarose gel showing endpoint PCR products obtained with DNA from HeLa-superT cells expressing TS or luciferase sgRNA harvested at the indicated times after AdCas9 infection. A plasmid template simulating neotelomere addition in the expected frame of addition was spiked into human DNA as a positive control (lane 5). (C) TaqMan-qPCR quantification of neotelomeres in (B). (D) EthBr-stained agarose gel showing endpoint PCR products obtained with DNA from HeLa-superT cells treated with BIBR1532 (20 μM) or DMSO (vehicle). Sample labeling and controls as in (B). (E) TaqMan PCR quantification of neotelomeres in cells in (D). Data points bearing the same color belong to the same biological replicate. (F) EthBr-stained polyacrylamide gel showing TRAP assay products obtained with extracts from p53^−/− Rb^−/− RPE1 cells infected with pLenti-sgTS-TaqMan-TS and then infected with a retroviral vector (vec), a retrovirus expressing catalytically dead (CD) hTERT, or a retrovirus expressing wild-type hTERT plus hTR (superT). As a control, extracts were heat inactivated (heated). IC, internal PCR control. (G) Quantification of neotelomeres formed in the RPE1 cells shown in (F) at the indicated times after infection with AdCas9. sT, superT. Mean ± SD of at least 3 biological replicates. * p < 0.05, ** p < 0.01, *** p < 0.001, two-tailed ratio-paired t-test in (E) and two-tailed unpaired t-test in (G).