Team:Nanjing NFLS/Experiment
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General info of Tage System
Tage system, the telomerase-activated gene expression system, is a versatile platform that acts like a missile which carries the effector gene “warhead.” The “warhead” of the system is changeable, as in this project we utilize Cas9 as the warhead to cut the telomeres of cancer cells and induce their apoptosis. But, based on such system, the "warhead" could be other proteins or microRNAs— ZsGreen was employed as the reporter gene "warhead" in the testing of specificity of Tage system.
The vector that carries the "warhead" is named based on its structure. Below are the examples of sTMEP and sTMCP. More details about each part of the vector can be seen in respective DNA design sections.
Summary
We first evaluated the specificity of Tage system which include the sTMEP we constructed; then we verified the effectiveness of Tage system using sTMCP. In order to make possible future in vivo application, we designed and tested a new Tage system consisted of C1-HO, HOsite-bTMEP, TsgRNA and dCas9-VP64 after experimentally showing that the 4-bp overhang produced by HO enzyme cutting its target DNA can be used by Tage system in human cells. At last, we simplified the system by synthesizing a coexpression plasmid TsgRNA-dCas9-VP64.
Evaluation of the specificity of Tage system
DNA Design— ZsGreen as the effector gene
In this part, we employed ZsGreen, the gene that codes for EGFP (Enhanced Green Fluorescent Protein) as the reporter gene (Fig.3-1) to test whether the system targets only cancer cells (Fig.3-2). The vector consists of a stick-end telomerase recognition sequence, a minimal promoter, the ZsGreen reporter gene and poly(A).
Is the system activated in and only in cancer cells?
We verified the specificity of the Tage system with a reporter construct. The Tage system includes an effector that has ZsGreen as the reporter gene (sTMEP). Both telomerase-active cells (HepG2) and telomerase-negative cells (HL7702) were transfected with sTMEP, TsgRNA and dCas9-VP64. The cells were observed and photographed with a fluorescence microscope (Olympus) at a constant magnification of 200×. The fluorescence intensity of cells were quantified with a flow cytometry (Calibur, BD, USA) and the mean fluorescent intensity (MFI) was calculated by BD software of flow cytometry.
The results (Fig.3-3) indicate that the ZsGreen protein was expressed by HepG2, in which telomere activity is present (Fig. 3); ZsGreen protein was not expressed by HL7702, which are cells with negative telomerase activity (Fig. 3). These results reveal that the Tage system can only be activated by telomerase in cancer cells.
Also, these results (Fig.3-3) suggest that the newly synthesized telomeric DNA sequence at the end of effector was double-stranded, since dCas9/TsgRNA only bind double-stranded DNA.
The fluorescence microscope pictures and representative flow cytometry analysis of HepG2, and HL7702 cells, which were transfected by a Tage system with an reporter gene ZsGreen.
Killing cancer cells with Tage system
DNA Design— Cas9 as the effector gene
To kill cancer cells in this part, we constructed the vector with Cas9 as the effector gene (Fig.4-1, Fig.4-2). The vector consists of a stick-end telomerase recognition sequence, a minimal promoter, the Cas9 reporter gene and poly(A).
Does the system kill cancer cells and only cancer cells?
We would like to kill cancer cells by cutting off their telomeres with a Tage system that has effector gene Cas9.
We conceived that the TsgRNA guides the Cas9 protein to the telomeres on chromosomes, where Cas9/TsgRNA cuts telomeric DNA and induces cell apoptosis. To verify this process, we transfected cells (293T, HepG2, HeLa, A549, Hepa1-6, HL7702) with a Cas9-expressing effector (sTMCP) together with vectors expressing TsgRNA and dCas9-VP64, and cultivated the cells for 24h. Then cells were washed and stained with Acridine orange, observed and photographed under fluorescent microscope (Olympus) at constant magnification of 200×.
The results show that the Tage system can induce significant death of all transfected cancer cells, including HepG2, HeLa, A549, and Hepa1-6, while not causing the apoptosis of normal cells HL7702 (Fig. 4-3).
The microscope pictures and representative acridine orange staining pictures of various cell lines transfected by the Tage system. The rate of cell decease is significant in HepG2, HeLa, A549, and Hepa1-6 but not in HL7702 cells.
Upgrading Tage system for future in vivo application
All of our experiments above were done in vitro, which means our linear effectors with a telomerase recognizable stick end could be easily transferred into cells by lipofectin. However, for this cancer therapy to be used in vivo in the future, the effector has to be transfected using virus vectors, which are incompatible with the stick end. So, we sought to address this issue. Through extensive research under the instructions of the PI, we found out that when cutting its target DNA, Homothallic switching endonuclease (HO) produces a four-base overhang (3’-TTGT-5’) that can be recognized and elongated by telomerase in S. cerevisiae cells.
Can 3’-TTFT-5’ be used in human cells?
To evaluate if this 4-bp overhang can be recognized in human cells, we constructed an effector that can express ZsGreen with a stick end of 3’-TTGT-5’ (HOsite-sTMEP). We transfected it into cells with TsgRNA and dCas9-VP64.
The results we obtained show that ZsGreen was expressed in HepG2 where telomerase activity is present, but not in normal cell HL7702 (Fig.5). These results indicate that the overhang can be used in the Tage system.
Having verified that 3’-TTGT-5’ can be used in the Tage system, we explored if the expressed HO protein can cut a blunt-ended effector with HO-site (HOsite-bTMEP) to produce the stick end of 3’-TTGT-5’. We constructed another vector coding for HO protein (C1-HO) and made a new Tage system with C1-HO, HOsite-bTMEP (the blunt-end effector), TsgRNA and dCas9-VP64. The results of cell transfections reveal that ZsGreen was expressed by telomerase-positive cells HepG2 but not by normal cell HL7702 (Fig.5), which indicates that HO protein cut the blunt end effector, producing a stick end.
The fluorescence microscope pictures of HepG2 cell lines and HL7702 cell lines.
Simplification of the system
Coexpression plasmid— TsgRNA-dCas9-VP64
We constructed a TsgRNA and dCas9-VP64 coexpression plasmid (TsgRNA-dCas9-VP64), and confirmed the plasmid by cotransfecting it with C1-HO and T-HOsite-bTMCP. The results reveal that this Tage system including a plasmid can cause significant death in HepG2 cancer cells but not in normal cells MRC-5 and HL7702 (Fig. 6).
The microscope pictures and representative acridine orange staining pictures of HepG2 cell lines and HL7702 cell lines. Significant death caused in HepG2 cells.
With the three vectors (TsgRNA-dCas9-VP64, T-HOsite-TMCP and C1-HO), it becomes possible to apply the Tage system in vivo using the virus vector.
Reference
S. J. Diede, D. E. Gottschling, Cell 99, 723 (1999).