Fig. 3. Telomerase forms functional neotelomeres.

(A) Experimental strategy to select for cells in which telomerase has formed a functional telomere at a DSB. A schematic of the CRISPR/Cas12a-edited chromosome 16 in the HeLa-superT 16p targeted clone (16p-targ) is shown, indicating the position of the knock-in cassette (grey) between FAM234A and LUC7L and the approximate locations of the 16q (green) and 16p (red) FISH probes. The knock-in cassette contains two homology arms and a Cas9-cleavable TS site separated by 500 bp bacteriophage λ DNA from the herpes simplex virus (HSV) thymidine kinase (TK) gene, which sensitizes cells to ganciclovir (Ganc). Positions of PvuII restriction sites and the 904-bp Southern blotting probe are indicated. Functional neotelomere formation at TS is predicted to result in loss of TK expression, ganciclovir resistance, loss of the 16p FISH signal, and a change in the size of the PvuII restriction fragment. ψ, MMLV packaging signal; CMV, cytomegalovirus promoter; P2A, 2A peptide from porcine teschovirus-1; bsd, blasticidin S deaminase gene; SV40-pA, simian virus 40 polyadenylation signal. The inset shows the sequence of TS PCR cassette, with the primer and TaqMan binding sites and patient-derived TS sequence indicated. (B) Representative examples of metaphase FISH (see (A)) performed on the parental HeLa-superT 16p-targ and one of the Ganc-R daughter clones (clone 13). The three copies of chromosome 16 are numbered and shown at higher magnification below. (C) Quantification of metaphase FISH in (B). Copies of chromosome 16 were identified by hybridization with either the p or q arm probe and scored as hybridizing with both the p and q arm probes, only the p arm probe, or only the q arm probe. Mean ± SD from eight technical replicates of 23 metaphases for the HeLa-superT 16p-targ and pooled data from clones 13, 34, 50, 54, and 64 (1-3 replicates per clone with 18-27 metaphases per replicate). ns p > 0.05, **** p < 0.0001, two-tailed unpaired t-test. (D) FISH on clone 13 with the two chromosome 16 probes in combination with a telomere probe (yellow). The partial metaphase shows one intact chr. 16, an irrelevant autosome, and the truncated chr. 16. (E) Southern blot of PvuII-digested DNA from the parental HeLa-superT 16p-targ and nine Ganc-R neotelomere clones probed with the 16p cassette probe indicated in (A). The position of the neotelomeres is indicated. (F) Southern blot of DNA from the indicated clones treated with Bal31 exonuclease as indicated, followed by digestion with PvuII. Left: EthBr-stained gel; middle: blot hybridized as in (E); right: same blot reprobed with a radio-labelled telomeric C-strand oligonucleotide.