Template:CasX

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.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 360
  
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 1552
  Illegal NgoMIV site found at 1825
  Illegal NgoMIV site found at 1984
  Illegal NgoMIV site found at 2437
  Illegal NgoMIV site found at 2494
  Illegal NgoMIV site found at 3367
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 58
  Illegal SapI.rc site found at 3362