CRISPR-Cas13a Multiplex Cancer Diagnostics via Lateral Flow
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Solution Overview
Problem
Current nucleic acid detection methods face challenges in achieving high sensitivity and specificity while being cost-effective and portable, particularly in point-of-care settings for cancer diagnostics.
Innovation Solution
A CRISPR-based nucleic acid detection system utilizing optimized guide molecules and RNA-based masking constructs, including ribozymes and aptamers, to detect cancer-specific fusion genes with high sensitivity and specificity, using lateral flow devices and amplification reagents like RT-RPA, enabling rapid detection within 45 minutes to 3 hours.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If qPCR approaches are used for nucleic acid detection, then sensitivity is improved, but device complexity and cost increase due to expensive instrumentation and complex operation requirements
Solution Approach 1:
The patent replaces complex mechanical/qPCR instrumentation with a CRISPR-based biochemical detection system that uses Cas13a enzyme and guide RNA to specifically bind and cleave target nucleic acid sequences, producing detectable signals through simpler chemical reactions rather than complex mechanical amplification and detection equipment
Solution Approach 2:
The patent introduces guide RNA as an intermediary molecule that mediates between the Cas13a enzyme and the target nucleic acid, enabling specific detection through programmed RNA-DNA hybridization and enzymatic cleavage, thereby simplifying the detection system while maintaining high sensitivity
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 optimizes reaction parameters including temperature (maintaining isothermal conditions at 37°C), pH, and buffer composition to maximize Cas13a enzymatic activity and guide RNA binding efficiency, thereby achieving high sensitivity detection on portable platforms without requiring complex thermal cycling equipment
Solution Approach 2:
The patent performs preliminary sample preparation steps including RNA extraction and concentration optimization before the CRISPR detection reaction, ensuring that the input material is of sufficient quality and quantity to enable sensitive detection on portable platforms with limited processing capabilities
3Measurement precision
If CRISPR-based detection systems are used to achieve high sensitivity and specificity, then detection precision is improved, but manufacturing complexity increases due to optimized guide molecule design and RNA-based masking constructs
Solution Approach 1:
The patent designs a universal CRISPR detection platform where the Cas13a enzyme and basic reaction components remain constant, while only the guide RNA sequences need to be changed to detect different target sequences, thereby simplifying manufacturing and enabling multiplexed detection with a single optimized platform
Solution Approach 2:
The patent divides the detection system into modular components including the Cas13a enzyme, guide RNA molecules with specific spacer sequences, and RNA-based masking constructs, allowing independent optimization and manufacturing of each component while maintaining overall system specificity and sensitivity
4Adaptability or versatility
If multiplex detection of multiple cancer fusion genes is implemented, then diagnostic versatility is improved, but device complexity increases due to multiple detection constructs and masking constructs
Solution Approach 1:
The patent creates a universal detection platform where a single CRISPR-Cas13a system can detect multiple different cancer fusion genes by simply changing the guide RNA sequences, allowing multiplexed detection of various cancer types and fusion genes without requiring separate detection systems for each target
Solution Approach 2:
The patent implements nested detection constructs where RNA-based masking constructs contain embedded guide RNA sequences that are sequentially activated, allowing multiple detection reactions to occur within a single integrated system while maintaining manageable complexity through hierarchical organization
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
The system allows for simultaneous detection of multiple cancer fusion genes with high sensitivity (down to 2 fM) on a multiplex lateral flow strip, providing a rapid, cost-effective, and portable solution for cancer diagnostics.
Implementation Method 1
one or more optimized guide molecules designed to bind to one or more corresponding target molecules of one or more cancer fusion genes
Implementation Method 2
the RNA-based masking construct is a ribozyme that generates the negative detectable signal, and wherein the positive detectable signal is generated when the ribozyme is deactivated. In an aspect, the ribozyme converts a substrate to a first color and wherein the substrate converts to a second color when the ribozyme is deactivated
Implementation Method 3
The RNA-based masking construct, in embodiments, is an RNA aptamer and/or comprises an RNA-tethered inhibitor. In an aspect, the aptamer or RNA-tethered inhibitor sequesters an enzyme, wherein the enzyme generates a detectable signal upon release from the aptamer or RNA tethered inhibitor by acting upon a substrate
Implementation Method 4
using lateral flow devices and amplification reagents like RT-RPA, enabling rapid detection within 45 minutes to 3 hours
Data Source
AI summary
Systems and methods for rapid diagnostics related to the use of CRISPR effector systems and optimized guide sequences, including multiplex lateral flow diagnostic devices and methods of use, including for detection of cancer markers, are provided.