Wouldn’t it be great if we could insert a bit of programmable DNA into an organism’s genome and then modify that DNA after the fact to contain whatever instructions we wished? Well, we can’t quite do that yet. But we may be one step closer to that goal, thanks to the work of Jerome Bonnet, Pakpoom Subsoontorn and Drew Endy of Stanford University. They have designed what they refer to as ‘rewriteable recombinase addressable data' (RAD) module.
In essence, this is a cartridge of DNA that contains one set of instructions when inserted in one direction, but at different set of instructions when inverted. At any point, the researchers can determine which instructions are given to the cell (in this case E. coli) by controlling the orientation of that cartridge.
The researchers relied on the fact that some types of bacteriophages (also called phages), have perfected the art of shuttling their own DNA in and out of their hosts’ genomes. These are viruses that only attack bacteria. They use enzymes (integrases) to insert their genomes into that of the host cell at strategic locations. When the viruses are ready to leave the cell, other enzymes (excisionases) clip out the viral DNA and package it into protein coats.
The RAD module includes the genes for an integrase and an excisionase. Under some conditions only integrase is made and under others both are made. When integrase alone is produced, the module is inserted in one direction. When both are produced, the module is snipped out, inverted and reinserted in the opposite orientation. Because the RAD module also encodes fluorescent reporter proteins, the orientation of the module can be monitored by observing whether the cell glows red or green.
To be clear, once inserted, the RAD module can’t be rewritten to include any new instructions. It only contains whatever nucleotides it started with. In this case, it contained the instructions to glow red or to glow green. However, the researchers were able to control which of those two instructions the cell followed. After some fine tuning to prevent spontaneous flipping, they were able to reliably switch back and forth between those two sets of instructions.