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Target4_CasX

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). 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 cleaves DNA strands in a sticky-ended fashion; in CasX enzymes comprise a distinct family of RNA-guided genome editors (2019) this cutsite is characterized as 10 nt with cleavage site of 12-14 on the target strand, and 22-25nt on the non-target strand. In Position of Deltaproteobacteria Cas12e nuclease cleavage sites depends on spacer length of guide RNA (Selkova et al, 2020), this cutsite is later recharacterized as 18nt target strand and 22nt on the nontarget strand. The length of the spacer region on the target strand is 20nt. The target strand must be flanked by a TTCN PAM 5’ to the spacer region.

Engineering

In strand displacement reactions toeholds have a minimum length of 4nt and an optimal length of 5-7nt for optimal kinetics. Given that the sticky end of CasX is at least 4nt in length, it functions as a toehold to catalyze a toehold-mediated strand displacement reaction and generate fluorescent signals. GC base pairs are preferred for the short 4nt toehold as they allow stronger binding and therefore faster strand displacement reactions leading to fluorescence. Structure of the native target DNA of CasX compared to engineered targets are shown in order (1st dsDNA corresponds to Target 1 and so forth).

The products of cleavage go on to participate in a downstream strand displacement reaction with reporter gates and trigger strands. Schematic shown below.

Characterization and Verification

Annealing of strand complexes is confirmed by native PAGE gel. dsDNA fragments are shown to be at correct lengths:

Cleavage assay results show some low levels of cleavage of target strands for T1 and T2:

Gel annotation: A. Upshifted band, which shows the CasX protein in complex with dsDNA strands. Present on all lanes except the negative control, faintly. B. Downshifted band which is a product of the cleavage reaction with the dsDNA target strand. C. Downshifted band at 20bp (smeared) which is the reporter gate fragment generated from cleavage reaction.

Sequence and Features