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Revision as of 15:55, 12 September 2024
vanAB_tracrRNA_CasX
Usage and Biology
CasX is a broad name for a family of Cas proteins later reclassified under Cas12e. McGill iGEM uses DpbCas12e isolated from Deltaproteobacteria due to high in-vitro cleavage activity (variant PlmCas12e displays low activity in-vitro). CasX uses a dual-RNA guide effector composed of a tracrRNA and crRNA. The tracrRNA and crRNA guide complex contains a scaffold stem containing a bubble, an extended stem, and a triplex region.
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 the following guideRNA engineering conditions:
Characterization and Verification
We test the function of the enzyme under two conditions: The enzyme does in fact cleave the target DNA strand, which is verified by a native PAGE gel. The enzyme is able to cleave the DNA strand which triggers the cascade that causes fluorescent readouts.
We annealed the dsDNA templates for the tracrRNAs and crRNAs in 1:1 stoichiometric ratio in 1xTE/10x Mg2++ (final concentration 12.5mM). The dsDNA templates carried the T7 promoter for RNA transcription. dsDNA templates and target strands verified by DNA-PAGE. The RNAs were transcribed overnight and then purified, then verified by denaturing UREA-PAGE. However, upon purification, we were unable to get a successful RNA- denaturing gel, as no RNA was detectable on our gel.
To verify whether or not the T7 transcription reaction was successful, or if an issue in purification was the cause of the missing RNA, we ran a denaturing urea-PAGE following the T7 transcription (unpurified, after 1 hours of reaction time). We were able to verify the presence of the RNA. tracrRNA names are shortened.
Above: T7 transcription reaction in progress. dsDNA template can be seen as upshifted band (at 90nt for tracrRNA, and 67nt for most crRNA with the exception of 5' ext, 3' ext, 3' hairpin). RNA visible at expected lengths for most samples. Smears, bands at lower sizes are anticipated to be rNTPs and incomplete RNA transcription reactions.
We theorize the purification kit that was used (Biobasic RNA cleanup) was not properly optimized for the shorter RNA transcript length of our guides–45 and 78nt for the crRNA and tracrRNA respectively, with the exception of 5' extension, 3' extension, and 3' ds hairpin crRNAs. This caused the significant loss of most of our samples on our first attempts to transcribe and purify our RNA.