Page 70 - Biennial Report 2018-20 Jun 2021

GENOME EDITING APPLIED TO TELOMERASE REACTIVATION IN AGGRESSIVE
CANCER PROGRESSION

In more than 80% of human tumors up-regulation or reactivation of the telomerase enzyme
results in aggressive cancers. Multiple studies report a number of mutations within the
telomerase gene promoter in several cancer types. How these mutations reactivate telomerase
is poorly understood. Overarching aim of this proposal was to understand how clinically detected
mutations in aggressive cancers reactivate telomerase. Specifically, by application of
CRISPR/CAS9- mediated gene editing techniques it was proposed to create cellular models to
study in detail mechanisms that govern how clinically detected mutations affect telomerase
expression/activity in aggressive cancers.

It was hypothesized that the hTERT promoter could be a fulcrum for interaction between distant
regions of the genome and promoter mutations could lead to disruption of these genome wide
looping interactions. Moreover, in vitro studies from the group along with others have shown
the potential of clinically prevalent point mutations in the hTERT core promoter in de-regulating
telomerase expression. The aim was to understand the cross talk between long range chromatin
interactions with promoter mutations and telomerase re-activation in cancer initiation. A recent
report by the group demonstrated, telomere looping independent genome wide gene
regulation/epigenetic alterations occur in a telomere length dependent manner. This is
mediated by the partitioning of telomere repeat binding factor 2 (TRF2) between telomere ends
and targeted gene promoters. With the knowledge of this phenomenon, it becomes inevitable
to explore both telomere looping as well as non-looping aspects together.

Therefore, to study this cross talk it was required to develop a model of hTERT wild type and
mutant promoter cell lines. While looping studies indicated looping to occur only up-to a 10Mb
distance from nearest telomere end, non-looping mediated regulation was demonstrated to
occur more than 60Mbs away from telomeres. To delink the hTERT – transcription regulation
mediated by telomere looping vs telomere looping independent regulation, and how promoter
mutations induce telomerase de-regulation via both of these mechanisms, hTERT wild type and
mutant and promoter were inserted much beyond 10Mb from telomere ends. Experiments were
initiated to insert hTERT promoter using CRISPR/Cas9 technology at CCR5 locus that is >40Mb
away from telomere ends. For this process, published guide RNA targeting CCR5 locus from a
recent report was used. This guide RNA was cloned into a widely used spCas9 vector (pspCas9
(BB)-2A-Puro (PX459) V2.0. The sequence of guide RNA is 5’-GGAGAGCTTGGCTCTGTTGGGGG-3’
(reverse strand: chr3:46,372,669- 46,372,691/hg38). The guide RNA was cloned into the spCas9
vector using BbsI restriction enzyme. Successful cloning was confirmed by sequencing the
plasmid. Next, the donor vector was prepared using the plasmid AY10_pS. hTERT 1300 bp
promoter was cloned along with a Gaussia luciferase gene downstream of it, which was used to
experimentally determine the regulatory effects on the hTERT promoter activity. The Mlu1 site
in between the 2 homology arms was used to insert a MCS and clone TERT promoter driven
Gaussia luciferase. This successful cloning was confirmed as well by sequencing. Following this,
further experiments to insert the 1300bp hTERT promoter-Gaussia luciferase gene at CCR5 locus
in HEK293T cell line have now been initiated.

The focus was on TRF2, a telomere binding protein shown to have extra-telomeric occupancy
both via telomere looping dependent and independent ways. From TRF2 ChIP-seq data

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