Difference between revisions of "Part:BBa K5099017"

Latest revision as of 15:52, 21 September 2024

vanAB_tracrRNA_CasX

This tracrRNA codes for a complete reconstitution of the CasX guideRNA from the antimicrobial resistance gene against vancomycin.

Project Description

The Nucle.io project aims to provide rapid, point-of-care diagnostics that undercut current wait times for the return and analysis of laboratory test results such as blood culture (3 days) and PCR (1 day), by performing both diagnostic amplification and result computation in one reaction. This allows clinical decision making to happen on a faster timescale in emergency medical settings where time is of the essence. Sepsis is a disease responsible for 20% of global deaths. Each hour of delayed treatment leads to an 8% increase in mortality. When infection is treated early with accurate antibiotics, downstream complications such as organ failure can be prevented. The Nucle.io diagnostic uses a modular approach to achieve both accuracy and speed, leveraging the CasX protein to perform mRNA-based detection or a strand displacement cascade to perform mRNA amplification. The downstream module (computation) applies the Winner-Take-All neural network (WTA NN), which is a DNA computing architecture using toehold-mediated strand displacement reactions to analyze profiles of nucleic acids developed in Neural network computation with DNA strand displacement cascades (Qian et. al, 2011).

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.