iGEM Freiburg 2014

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Abstract

Optogenetics, a novel technology that allows temporal and spatial induction of gene expression by the use of light, is of growing importance for fundamental research and clinical applications. However, its biggest limitation is the time consuming introduction of transgenes into organisms or cell lines. In contrast, easy but unspecific gene delivery can be achieved by viral vectors. We, the iGEM Team Freiburg 2014, combine the advantages of both approaches – the temporal and spatial resolution of optogenetics, and the simplicity of gene transfer offered by viruses. To this end we designed a system where the entry of a virus is enabled or prevented by exposing the target cells to light of distinct wavelengths.

The ecotropic murine leukemia virus (MuLV) is a retroviral vector which enters a cell by binding to the cationic amino acid transporter (CAT-1). CAT-1 is present in cells of all mammals, but displays a high variability between different species in the third extracellular loop, which is the recognition sequence for the virus. Therefore, even close relatives of mice, e.g. rats or hamsters, are immune to the MuLV, but upon exogenous expression of murine CAT-1, cell lines from these species can also be infected. Hence we use the popular Chinese hamster ovary cell line CHO in our experiments.

For optogenetic targeting of specific subsets of cells, we employ three different systems: a red/far-red light system and a UVB light system based on proteins from the plant model organism Arabidopsis thaliana as well as a light-oxygen-voltage system from the marine bacterium Erythrobacter litoralis. In all three light systems gene expression is induced by recruitment of engineered transcription factors to DNA.

Without illumination the cells are in a dormant state and cannot be infected by the viral vector. Upon exposure to the appropriate wavelength, they start expressing the viral entry receptor CAT-1. Addition of the viral vector to the culture medium leads to infection of the activated subset of cells. In order to demonstrate the functionality of the specific gene delivery we developed a device for secure communication. The device is working in a two-step process: First, the sender has to specify the subset of cells by illumination through a patterned mask. In the second step, upon receiving the device the reader has to visualize the message by adding the appropriate viral vector containing a reporter gene. The unintended opening of the device by an unaware third person results in a complete illumination of the cells leading to the destruction of the message. The time gap between sending and receiving the device is limited by the half-life of the receptor, thus creating an additional safety level. To expand into the field of medicine, we furthermore chose CRISPR/Cas as a gene cargo.

CRISPR/Cas has attracted much attention in recent past, as it facilitates specific gene editing, e.g. knock-outs/ins, with high specifity in a minimum of time. In combination with delivery by viral vectors this could provide a powerful tool for the treatment of diseases and genetic disorders in vivo.

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