CRISPR-Based Viral Detection via Isothermal Multiplexing
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Solution Overview
Problem
Current nucleic acid diagnostics lack the ability to perform massive multiplexing with high specificity and sensitivity at a low cost, limiting their application in clinical and basic research settings, especially for pan-viral or pan-pathogen testing.
Innovation Solution
The development of CRISPR-based diagnostic methods using RNA targeting effector proteins and guide RNAs to detect nucleic acids in droplets, enabling multiplexed reactions with small volume samples and distinguishing between single base pair differences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If qPCR approaches are used for sensitive detection, then detection sensitivity is improved, but cost increases and device complexity increases
Solution Approach 1:
The patent replaces complex qPCR instrumentation with a simpler isothermal amplification system using CRISPR-Cas12a or Cas13a enzymes. The mechanical/thermal cycling apparatus of qPCR is substituted with an isothermal reaction system that operates at constant temperature, eliminating the need for thermal cyclers while maintaining detection sensitivity through the collateral cleavage activity of Cas12a/Cas13a that amplifies signals without requiring complex instrumentation.
Solution Approach 2:
The patent changes the operational parameters from qPCR's temperature cycling (denaturation, annealing, extension at different temperatures) to isothermal conditions (constant temperature around 37-42°C). This parameter change simplifies the device requirements while maintaining detection sensitivity through the exponential amplification capability of the CRISPR-Cas system under isothermal conditions.
2Device complexity
If isothermal nucleic acid amplification with portable platforms is used, then device complexity is reduced for point-of-care settings, but detection sensitivity decreases
Solution Approach 1:
The patent incorporates a pre-amplification step using isothermal nucleic acid amplification (such as RPA or LAMP) before the CRISPR detection step. This preliminary action ensures sufficient target amplification to achieve high sensitivity, while the subsequent CRISPR-based detection maintains portability by using simple isothermal conditions and visual readout methods, thus resolving the contradiction between sensitivity and portability.
Solution Approach 2:
The patent introduces CRISPR-Cas12a or Cas13a as an intermediary between the isothermal amplification step and the final detection step. This intermediary enzyme provides signal amplification through collateral cleavage activity, enabling high sensitivity detection with portable platforms by bridging the gap between simple isothermal amplification and sensitive detection requirements.
3Productivity
If multiple pathogens are detected simultaneously, then productivity increases, but primer design complexity increases
Solution Approach 1:
The patent employs universal CRISPR detection components (Cas12a or Cas13a enzyme, guide RNA structure, collateral cleavage mechanism) that can detect multiple different pathogens. Instead of designing completely separate detection systems for each pathogen, the same core detection machinery is used with only the guide RNA sequence needing modification, thus enabling multiplexing while minimizing design complexity through the universal nature of the CRISPR system.
Solution Approach 2:
The patent segments the detection system into modular components: a universal CRISPR detection module (Cas enzyme, detection protocol, readout method) and pathogen-specific guide RNA sequences. This segmentation allows multiple pathogens to be detected simultaneously by simply changing the guide RNA segment while keeping the rest of the system unchanged, thereby increasing productivity without proportionally increasing overall system complexity.
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
This approach allows for robust, sensitive detection of DNA and RNA with high specificity, enabling multiplexed applications such as viral detection, bacterial strain typing, and disease-associated cell-free DNA detection, while reducing costs and increasing throughput.
Implementation Method 1
guide RNAs designed to bind to corresponding target molecules
Implementation Method 2
contacting a sample with a primer pair and a probe with a detectable label
Data Source
AI summary
Methods for generating primers and/or probes for use in analyzing a sample which may comprise a pathogen target sequence are provided, including identifying pan-viral sets of primers and/or probes.