By Asal Golshaie and Christian Donohoe
When the team first met, we only knew that we had ten weeks to use genetic engineering to “make something useful”. The rest was up to us. We bounced some ideas around, from synthetic honey to a treatment for gastric cancer. Eventually we settled on the idea of improving the diagnosis of sexually transmitted infections (STIs), alert to national cuts to sexual health spending and Cardiff University’s poor ranking for sexual healthcare.
Specifically, we set out to develop a novel point-of-care test for STIs: a faster, cheaper, and more specific alternative to sending samples for lab testing. A diagnostic test has two components: detection and reporting. For the detection of STI specific DNA, we turned to CRISPR-Cas9, a revolutionary gene editing tool based on the innate immune system of bacteria. For reporting, we relied on fluorescent proteins: they glow a certain colour when active, and are often fused to genes to follow their activity. We also evaluated the potential use of our system as a self-testing kit for home testing by investigating the ethical and social implications.
Popularly described as satnav and scissors, CRISPR-Cas9 is made up of guide RNAs (the satnav) that recognise a specific DNA sequence, and navigate the Cas9 (the scissors) to cut it. We removed the scissors from our design by using a dead Cas9 (dCas9) variant, which instead opens the DNA double helix when guided to the sequence. Upon the advice of Dr Patrick Hardinge, we used two single guide RNAs to increase the fidelity of the reaction, and in turn reduce false positives.
We chose firefly luciferase as our reporter since it produces green light that is visible to the naked eye (by using energy to catalyse the oxidation of its substrate, luciferin). It also didn’t hurt that one of our supervisors is a luciferase expert with plentiful access to a useful thermostable variant. In our system, we used split luciferases joined to separate dCAS9 enzymes. These split reporters are inactive until they recombine- this only happens if two guide RNAs target the dCas9s to the STI specific DNA sequence. Simply put, if the STI specific DNA is detected, the guide RNAs rejoin the dCas9 fused split luciferase fragments, resulting in a green glow.
We achieved limited success with this very ambitious project- too ambitious for our small time window, especially with our inexperience and many DNA ordering misshaps. In the end, only our guides were successful, as our split-luciferase failed to clone correctly, meaning we couldn’t study the viability of our system. However, we still think it has great potential for future iGEM teams to build on.
Luckily we had a side project to fall back on to meet other medal requirements. This of course still had to be ‘useful’. Upon Dr Amit Jathoul’s suggestion, we turned towards improving tissue imaging while building on the work of the Cambridge iGEM 2010 team. We worked on engineering mkeima (a red fluorescent protein) into the widely used lux operon of marine bacteria, so it would produce red light instead of blue. Since red light penetrates tissues, this has the potential to be a valuable research tool. Once again, we had limited success, but alongside our successful aid to the projects of two teams, Oxford and Washington in St Louis, we pulled through and got a silver medal- a decent feat for an inaugral team!
Although not all our designs were full realised, we believe we have set a clear path for others to carry on our work, but more importantly gained the experience to effectively mentor next year’s team to achieve a Gold in Boston.
If you wish to be a part of next year’s team, please contact Dr Geraint Parry ParryG5@cf.ac.uk
For more info, about our project, please check out our wiki & twitter.