THESIS
2021
1 online resource (xv, 115 pages) : illustrations (chiefly color)
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. G
2 DNA checkpoint activation was found to limit mitotic entry. A small portion of cells was able
to proliferate with mitotic defects after a prolonged G
2 arrest, resulting in the accumulation of
chromosome instabilities. I further showed p...[
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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. G
2 DNA checkpoint activation was found to limit mitotic entry. A small portion of cells was able
to proliferate with mitotic defects after a prolonged G
2 arrest, resulting in the accumulation of
chromosome instabilities. I further showed pharmaceutical inhibition of WEE1, CHK1/2, or ATR could abrogate the G
2 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.
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