The antibiotic resistance crisis is an ongoing phenomenon whose consequences are far-reaching – with the WHO describing it as “one of the biggest threats to global health, food security and development today” . While naturally occurring, the overuse of antibiotics in a variety of ways has accelerated this process, as humanity and pathogenic bacteria are locked in an arms race in which the former has the disadvantage. The term “post-antibiotic era” had already been established as early as 2010 , but a way to end the crisis has remained elusive.
Carbapenem resistant Enterobacteriaceae (CRE), also known as Carbapenemase-producing Enterobacteriaceae (CPE) are a part of this phenomenon. As their name would suggest, these bacteria, including strains of species like Klebsiella pneumoniae and Escherichia coli, which have developed resistance mechanism to a family of antibiotics known as Carbapenems. Carbapenems in many cases are used as“last resort” antibiotics for the treatment of infections. Resistance to Carbapenems is often assessed through routine swabbing when patients are admitted into hospitals, but a positive result has a turnaround time on the scale of days.
Faster tests, such as the Carba NP test, exist, but they are not effective in detecting all the possible Carbapenem resistance mechanisms  , leaving open the possibility for false negatives. Patients who test positive for CRE are isolated, but the aforementioned long turnaround time and potential for false negatives can lead to a window of unchecked spread of Carbapenem resistance throughout a hospital ward.
Our team has designed a novel detection method based on riboswitch-guided CRISPR activation of the transcription of a reporter, which has been proven to work in an in vivo scenario and can be readily converted to an in vitro cell-free system.
We drew upon previously existing work in the field of CRISPR activation (CRISPRa), notably by past iGEM teams such as Warwick iGEM 2016 and 2018, for inspiration. This exposed us to the many possibilities for iteration and improvement. More importantly, they showed us how the CRISPRa system has a high degree of specificity. This would be extremely useful in our application as we wanted to detect a specific strain of Carbapenemase resistance. As a detection target, we settled upon a Carbapenemase known as OXA-48, due to the difficulty of its rapid detection, even with the Carba NP test, due to its slow activity.
We settled upon the design of a guide RNA (gRNA) which, under normal conditions, folds in such a way that the binding of the gRNA to a Cas9 protein is impossible. However, when the OXA-48 mRNA is present, the two RNAs anneal, causing the gRNA to adopt a conformation which allows the binding of gRNA to Cas9. Furthermore, the version of Cas9 we use is also modified. We used a deactivated form of it (dCas9), which retains its ability to bind the gRNA but lacks its nuclease activity. For activation, we selected the SoxS system, composed of a fusion between an RNA-binding protein and a transcriptional activator . This protein binds to a groove in the gRNA when it is bound to dCas9, stabilising any transcriptional complexes that might form downstream.
Our plasmids (fig. 1 and 2) contain all these components, as well as the gene encoding our reporter, a fluorescent RNA aptamer named iSpinach, which, in combination with a non-toxic fluorogen known as DFHBI, produces a strong green signal visible under UV light. The PAM sequence for the gRNA we engineered is located just upstream of the promoter of this gene. We chose to pursue the use of fluorescent RNAs since it eliminates the need to wait for translation to occur – healthcare professionals have mentioned that time is of the essence when CRE are suspected.
 WHO fact sheet at https://www.who.int/news-room/fact-sheets/detail/antibiotic-resistance, accessed on the 27th of September 2021
 Zucca, M., Savoia, D., The Post-Antibiotic Era: Promising Developments in the Therapy of Infectious Diseases, Int. J Biomed Sci (2010), 6(2), pp. 77-86
 Tijet, N., Boyd, D., Patel, S. N., Mulvey, M. R., Melano, R. G., Evaluation of the Carba NP Test for Rapid Detection of Carbapenemase-Producing Enterobacteriaceae and Pseudomonas aeruginosa, Antimicrob Agents Chemother (2013), 57(9), pp. 4578-4580
 Osterblad, M., Hakanen, A. J., Jalava, J., Evaluation of the Carba NP test for Carbapenemase Detection, Antimicrob Agents Chemother (2014), 58(12), pp. 7553-7556
 Dong, C., Fontana, J., Patel, A., Carothers, J. M., Zalatan, J. G., Syntheic CRISPR-Cas gene activators for transcriptional reprogramming in bacteria, Nat Commun (2018), 2489(9), doi: https://doi.org/10.1038/s41467-018-04901-6