Fig. 5: Threonine 249 in the endogenous hTERT locus is edited by CRISPR-Cas9 in 293T cells.

a Genome editing strategy to introduce alanine or glutamic acid substitution in the endogenous hTERT locus. Green letters denote the PAM (protospacer adjacent motif) sequence for genome editing by CRISPR-Cas9. b PCR fragments surrounding target regions (1,184 bp for T249A and 456 bp for T249E) were amplified from genomic DNA of genome-edited 293T clones. PCR fragments from pBABE-puro-hTERT (plasmid control), 293T control cells (Control-CRISPR) and mutant cells (T249A-CRISPR, T249E-CRISPR) were digested with the indicated restriction enzymes and electrophoresed in 5% polyacrylamide gels. The bands enclosed with square and indicated by arrows show that the cleavage patterns of PCR fragment were changed in CRISPR-mutant clones. c Sanger sequencing traces of a control and two mutant clones generated from gel-purified PCR fragments of the genomic DNAs of the respective clones. The sequences boxed in red and blue are the base triplet coding for alanine and glutamic acid in mutant clones, respectively. d IP-RdRP assay using hTERT proteins immunoprecipitated from 293T CRISPR clones (upper panel). The relative RdRP activities are noted below the panel. hTERT proteins isolated by immunoprecipitation from the cells were detected by anti-hTERT mAb (clone 2E4-2) (lower panel). e Tumors generated from 1 × 10⁵ cells of 293T Control-CRISPR or 293T-T249A-CRISPR in eight NOD/SCID mice for each are presented (upper panel). The volume of tumors from mice with Control-CRISPR (black line) or T249A-CRISPR (red line) is shown as the mean ± SEM (n = 8, p = 0.0469 by unpaired t-test, two-sided) (lower panel). f Direct telomerase assay using hTERT proteins immunoprecipitated from Control-CRISPR or T249A-CRISPR. The relative telomerase activities are noted below the panel (upper panel). Immune complexes were isolated from Control-CRISPR and T249A-CRISPR with anti-hTERT mAb (clone 10E9-2) and associated hTERC RNA were subjected to RT-PCR (lower panel). Experiments were repeated five times (for d, f) with similar results. Source data are provided in the Source Data file.