Development of genetic ‘monitors’ to detect when genes are active – a possible tool in the fight against malaria?
Dr Roberto Galizi of the Centre for Applied Entomology and Parasitology (CAEP) - Keele University and scientists from the University of Warwick have developed genetic sensors that could function as a lab tests or as monitoring systems in either test tubes or living cells. These microscopic molecular machines can detect when specific genes are activated, or introduced to the cell, and react dynamically to those changes to execute on-demand functions.
The technology is based on the CRISPR gene editing system, widely used by scientists to target and modify specific genomic sequences within cells as well as to alter their expression. To generate these novel genetic devices, the scientists used the ‘scaffold’ of the CRISPR guide RNA sequence (gRNA), which is the programmable part of CRISPR responsible for DNA targeting. The programmability of CRISPR allows it to be redirected to virtually any genetic target, such as specific genetic mutations that causes disease.
Details of these novel genetic sensors have been published in The CRISPR Journal* where the scientists demonstrate the CRISPR-gRNA could be reprogrammed, by introducing a sensor sequence, so that the CRISPR complex would bind the predesigned DNA target only after being activated by a specific trigger signal, such as short RNA segments of viral RNA sequences.
The authors tested the genetic devices both in cell-free reactions as well as in living Escherichia coli bacteria, by introducing a fluorescent gene that they could switch ‘on’ or ‘off’ only after interaction between the CRISPR sensing device and the triggering RNA molecule. They further validated their system to detect an RNA molecule derived from the HIV virus, exemplifying its potential usability in medicine.
The work is the first step in developing genetic devices that can sense precise genetic activity within a cell and execute specific genetic responses when activated by the specific molecular signal. The scientists believe their system will be useful for many researchers looking to program cells with greater sophistication, for example to generate new synthetic circuits, and could potentially be applied to a variety of biotech applications, including therapeutics and diagnostics.
Prof Alfonso Jaramillo, lead author from the School of Life Sciences at the University of Warwick explained the inovative approach to the work.
“This is quite different from gene editing, where you simply modify the genome. This is about watching the behaviour of the genome. If you have a monitor of the cell’s behaviour then you can make the cell correct that behaviour if you don’t like it, you can suppress it, or you can exploit that to switch on other genes.”
CAEP molecular biologist and co-lead author, Dr Roberto Galizi, enlarged on the therapeutic applications of the new technique.
“Coupling a genetic sensor with CRISPR tools offers an unprecedented opportunity for researchers to take genetic editing technologies to a completely new dimension. Eukaryotic cells could be programmed to detect deleterious mutations that may arise within its own genes, or to respond when invaded by pathogens like bacteria do naturally against phages.
One interesting feature is that we can program these molecular tools to sense any predesigned RNA molecule in a sequence-specific manner and, at the same time, target any desirable gene or genetic sequence to stimulate various genetic actions, all within the same cell.
Genetic technologies aimed to control vector-borne diseases could benefit from such innovation. For example, we could engineer mosquitoes to sense and counteract pathogen transmission, or deal with mutations that make vectors or pest insects resistant to insecticides.”
*‘Engineered RNA-Interacting CRISPR Guide RNAs for Genetic Sensing and Diagnostics’ will be published in The CRISPR Journal, DOI: 10.1089/crispr.2020.0029 Link: https://doi.org/10.1089/crispr.2020.0029