Part:BBa_K5099027

CasX_AMR_Device

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 Device

Mechanism

This device is composed of the CasX protein, the reengineered tracrRNAs, the custom dsDNA ligands, and the reporter gates that form the complete biologically engineered diagnostic. When the tracrRNA binds mRNA in solution, the guideRNA of CasX is reconstituted and the enzyme initiates a cis-cleavage event to cut a sticky end into double-stranded DNA. This "activates" the previously noncatalytic target strand, turning it into a seesaw gate on which DNA computing reactions can be completed. In our testing fo the device, we feed the CasX-generated target DNA strands directly into a fluorescent reporter gate. However, in downstream work, this can be generally interfaced with DNA computing architectures such as winner-take-all neural nets.

Validation of the individual parts that form this device–the CasX protein, dsDNA target gates, and tracrRNA reconstitution–can be found on the individual parts pages as linked under Sequence (composite part map). Additionally, you can navigate more of our documentation on the Contributions page of our wiki.