Using CRISPR Cas9 technology to disrupt TERT in telomerase as a cure for cancer

1. CRISPR/Cas9-Mediated TERT
Disruption in Cancer Cells
Wen et al. (2020), CRISPR/Cas9-Mediated TERT Disruption in Cancer Cells, International
Journal of Molecular Sciences, 2020, 21, 653, doi:10.3390/ijms21020653/
Group 2:
Fam Chi Ler (0343273)
Lee Xiao Wei (0343499)
Onn Sze Mun (0337881)
Tan Heng Lim (0342799)

2. Introduction
→ Cancer hallmark (>90%): activated TERT → high telomerase activity (Coblebatch et al,
2019)
Telomerase
TERT: repressed in most somatic cells
TERC: expressed in all cells
→ Other studies:
❏ Inhibiting GABPB1 needed for TERT transcription (Yuan et al, 2019)
❏ TERT peptide vaccines & direct oligonucleotide inhibitors (Guterres and Villanueva,
2020)
→ In this study:
Objective: To inactivate TERT using CRISPR/Cas9 as cure for cancer

3. CRISPR/Cas9
● Cas9 (nuclease) + gRNA = Cas9-guide RNA complex
● binding leads to double stranded breaks (DSB)
● DSBs can be repaired by HDR or NHEJ pathways
● Induces knockout / knockin mutations
● 3 gRNA (sg1, sg2, sg3) target TERT gene exon 2
(E2), exon 4 (E4) & exon 6 (E6)
● 2 gRNA (sg4 & sg5) target introns of exon 4 (E4)

4. Methodology
Cell culture
HELA, PANC1, SKBR3 were
supplemented with FBS and
cultured. HASMC were cultured
and Human iPSCs were cultured
in feeder-free conditions.
gRNA design
Guide RNAs were designed
based on the sequence of the
targeted locus using online tool
(CRISPOR)
Cell electroporation and
transfection
All cells (except PANC1 & SKBR3):
tube electroporation machine
PANC1 & SKBR3: Lipofectamine
transfection
- all transfected with spCas9 pDNAs
Determination of Indels and
E4 deletion
Indel rate: calculated after PCR
amplification of targeted regions
E4 deletion: dual gRNA Exon
Removal strategy by PCR
gRNA off-target
analysis
Each off target site was amplified
by specific PCR primers and
subjected to T7 endonuclease I
assay
Western blot
Loaded whole cell lysate onto
polyacrylamide gels, blocked
with milk, incubated with primary
antibodies, followed by
secondary antibodies
T/S ratio assay
Sybr Green quantitative PCR
was carried out and T/S ratio
was calculated by normalization
of telomere content with internal
control
Xenograft Experiment
Nude mouse legs were
inoculated to harvest Xenograpt.
Left hind leg: wild-type unedited
Hela cells
Right hind leg: gene-edited
TERT(+/-) Hela cell

5. Findings/Results
Indel efficiency
● Most efficient: Hela
● Least efficient: PANC1
Exon removal efficiency
● Most efficient: Hela
● Least efficient: PANC1
T/S ratio
● TERT+/− much lower than
WTPE

6. Findings/Results
Xenotransplant of WTPE and TERT+/−
.Hela cells in nude mice
● WTPE cells grew into tumours
● TERT+/- did not form any tumour
A: WTPE cells faster doubling time
B: WTPE cells higher density
C: TERT+/- higher apoptosis percentage
D: TERT+/- higher cellular death rate

7. Challenges/Limitation
❏ Lack of effective and safe delivery and substantial off target event
❏ Inhibiting GABPB1 needed for TERT transcription (Yuan et al, 2019)
❏ TERT peptide vaccines & direct oligonucleotide inhibitors (Guterres and Villanueva, 2020)
Solution
Identification and validation of tumor specific target gene(s)
-Development of efficient editing strategy on the target gene(s)
-Using RNAi (RNA interference) screens for target identification
About RNAi
-RNAi libraries were commonly used for screening gene function
-Can be used to studied where temporary loss-of-function is desired

8. Future developments
Removing gene SH-SY5Y in Telomerase can be a potential cure for Parkinson’s Disease (PD)
-After removal of telomere gene in SH-S5SY, there is a significant decrease (65%) in PD expression
-By using alpha synuclein, it can promotes DSB repair pathway, know as non-homologous end joining
(NHEJ) , which compromise in Lewy Bodies inclusion bearing neurons and may trigger cell death.
Limitations
- it causes mitochondrial dysfunction
-overload of alpha synuclein produce will cause alpha synuclein aggregation
Modification can be made
-Avoid damage on gene PINK1 which act as a protector for mitochondria from malfunctioning during
periods of cellular stress.

9. Conclusion
02 03
Identification &
development
Gene editing
Prerequisites
TERT gene
90% in all cancers
Identification
CRISPR-Cas9
gRNA targeting TERT
exon: up to 85%
efficacy
Development
01

10. References
Colebatch AJ, Dobrovic A, Cooper WA. (2019). ‘TERT gene: its function and dysregulation
in cancer’, Journal of Clinical Pathology, 2019;72:281-284.
Guterres, A.N., Villanueva, J. (2020). ‘Targeting telomerase for cancer therapy’.
Oncogene 39, 5811–5824 (2020). https://doi.org/10.1038/s41388-020-01405-w
Yuan, X., Larsson, C. & Xu, D. (2019). ‘Mechanisms underlying the activation of TERT
transcription and telomerase activity in human cancer: old actors and new players’.
Oncogene 38, 6172–6183 (2019). https://doi.org/10.1038/s41388-019-0872-9