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CRISPR cas9 mediated TERT disruption in cancer cells
- 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
Editor's Notes
- -cancer hallmark -chromosome-telomere-telomerase components -how does telomerase/TERT function in normal and in cancer → which is why tert is targeted for cancer treatment -other studies -this study (brief overview: method (gRNA design), results (KO TERT exon/efficacy for diff cancers), limitations and future developments) What is TERT? Why is it important? (Yuan et al 2019) Telomerase is a multi-unit complex, but its core enzyme is only composed of: TERT is a catalytic subunit of telomerase; TERC is internal telomerase RNA template ; telomerase repression and/or shorter telomeres in human cells function to prevent uncontrolled cellular proliferation TERT/telomerase activation has been experimentally shown to be essential to cellular immortalization and malignant transformation by stabilizing telomere length and erasing the senescence barrier. Consistently, TERT expression/telomerase activity is detectable in up to 90% of human primary cancers What is happening currently? (what are the current treatment targeting TERT)?Why TERT? What is the potential? Inhibiting GABPB1 (for TERT transcription) as glioblastoma treatment → increased invasiveness of cytotoxic T cells Typical examples that activate telomerase are myc/max/mad1 network protein. In addition, many other regulate TERT transcription, including ETS family.
- The targeting efficiencies of each gRNA, as determined by the insertion and/or deletion (indel) rates at the target locus, were evaluated by deep sequencing (deepseq). Sg1, sg2 and sg3 all worked well in Hela cells, with indel rates of 35.75%, 84.98% and 85.62%, respectively (Figure 1B). The same gRNAs (sg1, sg2 and sg3) led to lower indel rates in SUM159 cells (22.63 to 41.81%) and even lower in PANC1 cells (9.64 to 16.75%), indicating that the gRNA indel efficiencies may be cell line dependent. The cell proliferation, as measured by the population doubling time, was much slower in TERT+/− than that in WTPE Hela cells in culture (Figure 3A). Consistently, TERT+/− Hela cells were of lower density in culture than that of WTPE cells (Figure 3B,C). Cellular apoptosis enhanced in TERT as evidenced by Annxin and PI staining, Pierce LDH cytotoxicity assay also revealed higher cellular death rate in TERT.
- → Other studies: Yuan (2019) GABPB1 from ETS family of TERT transcription regulatory factors was inhibited. The cells depleted of GABPB1 suffered impaired survival, telomerase shortening, and attenuated tumorigene activity. BUT, in turn increased the invasiveness of cytotoxic T cells Guterres (2020) both TERT peptide vaccine (GV1001) and direct oligonucleotide inhibitor (imetelstat) demonstrated suppression of tumour growth in mouse models but neither approach demonstrated clinical efficacy.
- the present work worked on two prerequisites for gene editing-based cancer therapies: (i) identification and validation of tumor specific target gene(s); and (ii) development of efficient editing strategies on the target gene(s) TERT gene as the first choice to develop efficient gene editing strategies towards the goal of gene editing therapy for cancer. This is because tumor cells, up to 90% in all cancer types, activate TERT/telomerase to achieve immortality. As such, inhibiting TERT may provide a universal therapy for treating a wide spectrum of cancers Here we report several efficient gRNAs targeting the TERT gene exons or intros with indel rates up to 85%.