McGill iGEM hijacks mRNA in solution to force it to form the crRNA of the CasX system. An engineered tracrRNA binds to the crRNA to reconstitute the guide complex of CasX. This allows the enzyme to associate to the RNA and initiate a sequence-specific cleavage event upon a dsDNA target strand with a matching spacer motif that we place in excess in the solution. CasX cuts a sticky end at the 18-22nt region of dsDNA relative to the spacer. This sticky end is repurposed as a toehold in a strand-mediated displacement reaction, which allows the trigger of a fluorescent output upon mRNA detection through hijacking and cleavage of the target DNA.
We test the reengineerability of the tracrRNA through reconstitution of the guide complex, scaffold stem, and bubble of the triplex region with several guideRNA engineering conditions.
