Our system uses a mutated Cas9 protein from Streptococcus pyogenes to detect PCA3, a long non-coding RNA that works as a biomarker for prostate cancer detection; we engineered this Cas9 so that it produced a measurable signal as a result of the RNA detection.
Although our system was designed aiming to detect prostate cancer, it can be used to detect any DNA or RNA molecule. Some of the advantages our system has are:
Our system uses a modified dCas9 for detecting a specific nucleic acid molecule and the conformational change this protein suffers when it binds to the target is used to trigger a signaling mechanism. Since our dCas9 has intein insertions in two specific amino acid residues, the dCas9 conformational change provokes an intein reaction that enables the release of a split TEV. Next, following protein trans-splicing, TEV protease becomes catalytically active and it is able to cleavage a Green Fluorescent Protein from a Dark Quencher, which leads to fluorescence emission.
For a better understanding of our system, we organized it in three modules, which are described below:
Our detection system uses a modified dCas9 for detecting a specific RNA or DNA target.
Our signaling system is based on intein reactions that enable the releasing of a split TEV from our modified dCas9.
Our response system is based on the reconstitution of a split TEV, which is able to cleavage a Green Fluorescent Protein from its dark quencher, allowing fluorescence emission.