CRISPR/Cas9-mediated DNA damage to telomeres

Abstract

Telomeres are protective structures at the ends of eukaryotic chromosomes. Cancer can overcome the progressive shortening of telomeres in somatic cells through telomerase reactivation or alternative telomere maintenance to enable replicative immortality, suggesting that telomeres and telomerase are potential therapeutic targets. As CRISPR/Cas9 allows precise targeting, I studied whether targeting telomeres with CRISPR/Cas9 could be a therapeutic approach towards cancer. The effects of telomere CRISPR included cell cycle arrest and DNA damage response. G₂ DNA checkpoint activation was found to limit mitotic entry. A small portion of cells was able to proliferate with mitotic defects after a prolonged G₂ arrest, resulting in the accumulation of chromosome instabilities. I further showed pharmaceutical inhibition of WEE1, CHK1/2, or ATR could abrogate the G₂ DNA checkpoint and induced mitotic catastrophe. My experiments also demonstrated that telomere CRISPR led to extremely poor cell survival. These results suggest telomere CRISPR reduces cell viability through activation of DNA damage response and accumulation of chromosome instabilities. Telomere CRISPR was able to completely remove telomere signals. Furthermore, the removal of telomere was highly correlated with DNA damage at the chromosome level. I further confirmed the telomere length was reduced through a telomere length assay. Telomere CRISPR induced DNA damage in a dose-dependent manner. Chromosome fusion was also observed. Taken together, cell senescence and reduction of cell viability are induced after the removal of telomeres by telomere CRISPR. I further evaluated the sensitivity to telomere CRISPR in isogenic cell lines with different telomere number. More DNA damage was induced by telomere CRISPR in tetraploid cells than diploid cells. The cell fate was eventually the same at similar concentrations of telomere CRISPR. These results suggest the CRISPR/Cas9-mediated DNA damage to telomeres is likely to be saturated in cancer cell lines and further increase of telomeres do not affect the sensitivity.

Date of Award	2021
Original language	English
Awarding Institution	The Hong Kong University of Science and Technology
Supervisor	Randy Yat Choi POON (Supervisor)