Type III-A CRISPR Virus Detection for Dual RNA and DNA Sensing
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Solution Overview
Problem
Current CRISPR-Cas based diagnostic tools, particularly those using Class 2 systems like Cas12 and Cas13, are limited in their ability to simultaneously detect both viral DNA and RNA, making it challenging to repurpose Type III-A CRISPR-Cas systems for comprehensive virus detection due to complex enzyme reconstitution processes.
Innovation Solution
The development of MORIARTY, a Type III-A CRISPR-Cas system that harnesses dual nucleic acid cleavage activities through an in vivo-reconstituted effector complex, allowing for simultaneous detection of viral RNA and DNA without amplification or coupled to RNA transcription in a one-pot reaction, using L1Csm effector complexes and ancillary proteins like L1Csm6 to activate detectable DNase and RNase activities.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If Class 2 CRISPR-Cas systems (Cas12, Cas13) are used for virus detection, then detection of viral DNA or RNA can be achieved, but the ability to simultaneously detect both viral DNA and RNA is limited
Solution Approach 1:
The Type III-A CRISPR-Cas system is engineered to perform multiple detection functions simultaneously. The effector complex can detect both viral RNA through direct binding and viral DNA through transcription-mediated detection, making a single system universal for detecting both DNA and RNA viruses without requiring separate diagnostic tools for each nucleic acid type.
Solution Approach 2:
The Type III-A CRISPR-Cas system is divided into distinct functional modules: the effector complex (Csm1-Csm6) for target recognition and binding, the ancillary enzyme (Csm6 or Csx1) for collateral cleavage activity, and the reporter system for signal generation. This segmentation allows each component to be optimized for its specific function while working together to achieve simultaneous DNA and RNA detection.
2Adaptability or versatility
If Type III-A CRISPR-Cas systems are repurposed for comprehensive virus detection, then simultaneous detection of viral RNA and DNA is enabled, but the enzyme reconstitution process becomes complex
Solution Approach 1:
Multiple CRISPR-Cas subunits (Csm1, Csm3, Csm5) that were previously required as separate components are merged into a single polycistronic mRNA transcript. This allows the effector complex to be reconstituted from a single genetic template, dramatically simplifying the manufacturing and reconstitution process while maintaining the ability to detect both viral RNA and DNA.
Solution Approach 2:
The system is designed with a polycistronic mRNA that预先 (in advance) encodes all necessary effector complex subunits. This preliminary arrangement of genetic information allows the complex to self-assemble in vivo without requiring multiple separate transfection steps or complex in vitro reconstitution procedures, making the system easier to manufacture and deploy.
3Speed
If rapid virus detection is achieved within 30 minutes, then detection speed is improved, but detection sensitivity must be maintained at high levels
Solution Approach 1:
The system employs an ancillary enzyme (Csm6 or Csx1) as an intermediary that amplifies the detection signal. When the effector complex binds to the viral target, it activates the ancillary enzyme to perform collateral cleavage of reporter molecules. This intermediary mechanism provides signal amplification that maintains high detection sensitivity even within the rapid 30-minute detection window.
Solution Approach 2:
The system optimizes reaction parameters including temperature, pH, and component concentrations to achieve both rapid reaction kinetics and high sensitivity. By carefully tuning these parameters, the system accomplishes fast detection within 30 minutes while maintaining the ability to detect low concentrations of viral nucleic acids through enhanced signal amplification mechanisms.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
MORIARTY achieves high sensitivity with a detection limit of less than 500 fM, enabling rapid and cost-effective virus detection, including SARS-CoV-2, within 30 minutes, without the need for thermocyclers, and is adaptable to various buffer conditions for multipronged detection.
Implementation Method 1
CRISPR and CRISPR-Associated (Cas) proteins have been harnessed in nucleic acid detection owing to both their programmability and RNA-induced enzymatic activities
Implementation Method 2
The Cas13-based methods such as Specific High-sensitivity Enzymatic Reporter un-LOCKing (SHERLOCK) detect amplified viral RNA by monitoring cleaved RNA probe
Data Source
AI summary
Methods and systems, which use a reconstituted Type III-A CRISPR-Cas system, MORIARTY (Multipronged, One-pot, RNA Induced, Augmentable, Rapid, Test sYstem) for the detection of disease are provided herein. The methods and systems may be performed either without amplification or coupled to RNA transcription as one-pot reactions. The systems and methods herein may be highly sensitive and may be used to detect viruses, including SARS-CoV-2.