Investigating the Effects of Telomere Loss by CRISPR-Cas9

Telomeres, a type of nucleoprotein complex consisting of TTAGGG repeats and shelterin proteins found at the ends of chromosomes, are integral in maintaining genome integrity, which makes them highly relevant for stem cell and cancer cells’ proliferative capacity. Previous research has demonstrated that deprotection of telomeres by knocking down shelterin components leads to genome instability and telomere crisis, but the effect of direct manipulation of telomeric DNA on telomere function is less well understood. This study first demonstrated the efficacy of using a lentiviral CRISPR-Cas9 system targeting the telomere repeat in removing and shortening telomeres. We leveraged recent advances in long-read Nanopore sequencing to analyze changes in telomere length distributions with greater resolution than previously possible. Western blot and other Nanopore sequencing pipelines were used to characterize the cells’ response to telomere truncation. We found that this genome editing system induced drastic shortening of telomeres within a few days, leading to specific upregulation of non-homologous end-joining as well as genome instability. Additionally, Cas9-mediated telomere shortening temporarily slowed cellular proliferation with a crisis-like phenotype and induced TIFs. These data demonstrate the importance of intact telomeric DNA in maintaining the protective functions of telomeres to avoid genome instability and crisis. Characterizing the effects of telomere truncation would deepen our understanding of telomere biology and its interactions with other cellular pathways like the DNA damage response, senescence, and cell death, which could inform new modalities for cancer treatment.