Regulatory

Part:BBa_K1699001

Designed by: Emil Ruvinov   Group: iGEM15_BGU_ISRAEL   (2015-08-09)

Human short TERT promoter

Human short TERT promoter.

hTERT (human Telomerase Reverse Transcriptase) is a human promoter which controls the transcription of Telomerase, a gene highly expressed in cancer cells (1).


Usage and Biology

hTERT promotes the transcription of the catalytic subunit of telomerase. Telomerase elongates the ends of chromosomes, regions called telomers. hTERT is not active in somatic cells and is highly active in most if not all cancer cell types. Therefore, the expression of an exogenous gene under the control of hTERT promoter will likely occur in cancer cells only. We have used this promoter as part of a two promoter system (the other one being survivin promoter, also highly active in cancer cells), in order to drive the expression, and subsequently activate the core of our system (based on CRISPR?Cas9 technology) exclusively in cancer cells.

Characterization

The validity of hTERT promoter for the application of cancer-specific gene expression was performed using quantitative real-time PCR (qPCR), where the expression of hTERT was evaluated in several human cancer cell lines compared to healthy cells (fibroblasts) (Fig. 1). The results show thousand-fold and higher TERT expression levels in cancer cells, suggesting marked promoter hyperactivation.


Fig. 1. hTERT expression level in several human cancer cell types, evaluated by qPCR. HF-human fibroblasts (healthy cells); cancer cell lines: HepG2 - hepatocarcinoma, A549 - lung adenocarcinoma, MDA-MB-231 - breast adenocarcinoma, HT1080 - fibrosarcoma.


This part was used and validated by BGU 2015 team in a following constructs.
1. phTERT-GFP: AAV (adeno-associated virus)- vector expressing GFP under the control of human short TERT promoter was constructed (Fig. 2).


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Fig. 2. Plasmid map of AAV vector expressing eGFP under the control of human short TERT promoter.


Following calcium phosphate plasmid transfection or AAV transduction, GFP expression was evident only in cancer cells, compared to undetected levels in healthy cells (Fig. 3).


HTERT-GFP A.jpg
Fig. 3. eGFP expression under short human TERT promoter in cancer cells (fibrosarcoma) and healthy fibroblasts, after calcium phosphate plasmid transfection (A) or AAV transduction (B). Bar: 200 micron.



2. phTERT-dCas9-VP64: AAV vector expressing dCas9-VP64 (2) (engineered version of "classical" Cas9 for transcriptional activation of any gene of interest) under the control of human short TERT promoter was constructed as a part of functional prototype of two cancer-specific promoter-driven CRISPR/Cas9 activation system of BGU 2015 Boomerang team (Fig. 4).

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Fig. 4. Plasmid map of AAV vector expressing dCas9-VP64 under the control of human short TERT promoter.

Following simultaneous plasmid transfection of dCas9-VP64 - under the control of hTERT promoter, guide RNA (targeting the synthetic activation promoter) - under the control of human survivin promoter, and eGFP under synthetic activation promoter (3), eGFP expression was detected only in cancer cells, compared to undetected levels in healthy cells (Fig. 5). The unique ribozyme design was used to drive gRNA synthesis under "unusual" control of RNA polymerase II (and not III) promoter (4, 5).

Fig. 5. eGFP expression from synthetic activation promoter exclusively in cancer cells after successful activation of CRISPR-based activation core driven by dCas9-VP64 - under the control of hTERT promoter, and guide RNA (targeting the synthetic activation promoter) - under the control of human survivin promoter. Bar:100 micron.

The expression of dCas9-VP64 and gRNA under the control of cancer-specific promoters (TERT and survivin) drives the activation of the system only in cancer cells (Fig. 6).

Fig. 6. Cancer-specific CRISPR-based activation of the gene of interest using two cancer-specific promoter-driven expression of dCas9-VP64 and gRNA.


3. phTERT-SaCas9: We also engineered AAV construct in which the short human TERT controls the expression of "classical" Cas9 (SaCas9 (6)) (Fig. 7A). The construct, together with gRNA under the control of human survivin promoter link to registry part, could drive cancer-specific gene knockout (Fig. 7B).

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Fig. 7. A. Plasmid map of AAV vector expressing SaCas9 under the control of human short TERT promoter. B. Summary of two cancer-specific promoter-driven CRISPR-mediated gene knock-out.

References

1. The telomerase reverse transcriptase promoter drives efficacious tumor suicide gene therapy while preventing hepatotoxicity encountered with constitutive promoters. Majumdar AS, Hughes DE, Lichtsteiner SP, Wang Z, Lebkowski JS, Vasserot AP. Gene Ther. 2001 Apr;8(7):568-78.http://www.ncbi.nlm.nih.gov/pubmed/11319624

2. RNA-guided gene activation by CRISPR-Cas9-based transcription factors. Perez-Pinera P, Kocak DD, Vockley CM, Adler AF, Kabadi AM, Polstein LR, Thakore PI, Glass KA, Ousterout DG, Leong KW, Guilak F, Crawford GE, Reddy TE, Gersbach CA. Nat Methods. 2013 Oct;10(10):973-6. doi: 10.1038/nmeth.2600. Epub 2013 Jul 25. http://www.ncbi.nlm.nih.gov/pubmed/23892895

3. Tunable and multifunctional eukaryotic transcription factors based on CRISPR/Cas. Farzadfard F, Perli SD, Lu TK. ACS Synth Biol. 2013 Oct 18;2(10):604-13. doi: 10.1021/sb400081r. Epub 2013 Sep 11. http://www.ncbi.nlm.nih.gov/pubmed/23977949

4. Self-processing of ribozyme-flanked RNAs into guide RNAs in vitro and in vivo for CRISPR-mediated genome editing. Gao Y, Zhao Y. J Integr Plant Biol. 2014 Apr;56(4):343-9. doi: 10.1111/jipb.12152. Epub 2014 Mar 6. http://www.ncbi.nlm.nih.gov/pubmed/24373158

5. Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells. Nissim L, Perli SD, Fridkin A, Perez-Pinera P, Lu TK. Mol Cell. 2014 May 22;54(4):698-710. doi: 10.1016/j.molcel.2014.04.022. Epub 2014 May 15. http://www.ncbi.nlm.nih.gov/pubmed/24837679

6. In vivo genome editing using Staphylococcus aureus Cas9. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang F. Nature. 2015 Apr 9;520(7546):186-91. doi: 10.1038/nature14299. Epub 2015 Apr 1. http://www.ncbi.nlm.nih.gov/pubmed/25830891

Sequence and Features


Assembly Compatibility:
  • 10
    COMPATIBLE WITH RFC[10]
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    COMPATIBLE WITH RFC[21]
  • 23
    COMPATIBLE WITH RFC[23]
  • 25
    COMPATIBLE WITH RFC[25]
  • 1000
    COMPATIBLE WITH RFC[1000]


[edit]
Categories
//awards/basic_part/2015
//awards/part_collection/2015
Parameters
None