Immobilized CRISPR Assay for Multiplex Pathogen Detection
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Solution Overview
Problem
Current PCR-based assays for biosurveillance and diagnostics are time-consuming, taking weeks to months to develop and validate, and are limited in their ability to detect a broad range of pathogens in a user-friendly manner, especially when multiplexing multiple targets.
Innovation Solution
An assay method involving immobilization of guide RNA (gRNA) to a surface, complexing with Cas enzyme, adding target nucleic acid, labeling, and detecting without amplification, using a Cas sandwich protein with a randomized gRNA library to enhance specificity and sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR-based assays are used for pathogen detection, then detection capability is achieved, but development time takes weeks to months
Solution Approach 1:
The patent changes the fundamental detection parameter from PCR amplification to direct CRISPR-Cas binding detection. By using immobilized gRNA on surfaces and detecting target nucleic acids through specific Cas protein-gRNA-target complexes, the assay eliminates the time-consuming PCR amplification step while maintaining detection capability through high-specificity molecular recognition
Solution Approach 2:
The patent extracts and removes the amplification step from the detection workflow. By directly detecting target nucleic acids through CRISPR-Cas binding on immobilized surfaces, the method eliminates the intermediate PCR amplification process that traditionally takes weeks to months for development and validation
2Measurement precision
If traditional PCR assays are used, then pathogen detection is possible, but multiplexing multiple targets is limited
Solution Approach 1:
The patent segments the detection system into multiple independent gRNA-Cas protein complexes that can be simultaneously immobilized on different regions of a surface. Each gRNA is designed to recognize a specific target sequence, allowing multiple different targets to be detected in parallel through spatial segmentation of the assay surface
Solution Approach 2:
The patent creates a universal CRISPR-Cas detection platform that can detect multiple different pathogen targets using the same fundamental mechanism. By changing only the gRNA sequence while keeping the Cas protein and detection methodology constant, the system achieves multi-functionality for detecting various DNA and RNA targets
3Measurement precision
If PCR-based detection is used, then target nucleic acid can be detected, but assay development and validation take weeks to months
Solution Approach 1:
The patent replaces the complex mechanical and chemical amplification system of PCR with a simpler direct binding system based on CRISPR-Cas molecular recognition. This substitution eliminates the need for thermal cycling, polymerase enzymes, and amplification chemistry, dramatically reducing development complexity and time
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
Enables rapid and efficient detection of multiple pathogens without the need for pre-amplification, allowing for high multiplexing and rapid reconfiguration of pathogen detection panels, reducing development time to hours.
Implementation Method 1
immobilizing a guide RNA (gRNA) to an immobilization surface; complexing a Cas enzyme to the gRNA; adding a target nucleic acid
Implementation Method 2
complexing a Cas enzyme to the gRNA; detecting the target nucleic acid without amplification
Data Source
AI summary
The present disclosure relates to an assay method for detection of target nucleic acid. The method includes immobilizing a guide RNA (gRNA) to an immobilization surface; complexing a Cas enzyme to the gRNA; adding a target nucleic acid; labeling the target nucleic acid; detecting the target nucleic acid without amplification; and determining one or more results based on the detected target nucleic acid via a reader.