Background & Aims:
Telomeres are nucleoprotein structures that protect the ends of linear chromosomes. In human
somatic cells, telomeres shorten with each round of cell division, leading to replicative
senescence. Senescence can be overcome by telomerase, an enzyme specialising in the
maintenance of telomere ends by the synthesis of de novo telomeric repeats. Though typically
suppressed, the catalytic component of telomerase (TERT) becomes reactivated in the vast
majority of cancers to counteract telomere shortening and achieve proliferative immortality.
We aim to use human cancer cell lines to investigate the underlying mechanisms of telomerase
activation and repression during tumorigenesis.
Methods & Results:
We have used CRISPR/Cas9 to knock-in two versions of a bespoke reporter system at the
TERT 3’-UTR in the HCT116 cancer cell line. In the first version, the modified TERT locus
employs a P2A-T2A self-cleaving peptide to enable co-expression of TERT and EGFP driven
by the TERT promoter. The second version achieves co-expression using an Internal Ribosome
Entry Site (IRES) to segregate translation of TERT and EGFP. Clones with the reporter tags
successfully inserted have been isolated and confirmed by DNA sequencing. Telomerase
activity assays demonstrate that telomerase activity has been differentially affected by genome
targeting, while TERT, hTR, and EGFP gene expression can be detected by qRT-PCR. We are
in the process of optimising EGFP detection.
Conclusions:
We have created two novel reporter tag systems to detect and quantify endogenous levels of
TERT expression. We are currently developing the reporter systems for high throughput
applications in cancer cells with the aim of conducting genome-wide CRISPR screens to
investigate pathways of telomerase activation and repression that will ultimately further our
understanding of cancer cell immortalisation.