CRISPR Multiplex Nucleic Acid Detection via Cas Protein Segmentation
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Solution Overview
Problem
Current nucleic acid detection methods are limited in achieving rapid, simple, and accurate multiplex detection, which is essential for pathogen and genetic disease diagnostics.
Innovation Solution
A method and system utilizing CRISPR-associated (Cas) proteins, guide RNA, and single-stranded nucleic acid reporters to detect target nucleic acids, enabling multiplex detection through specific cleavage and generation of detectable signals for various nucleic acid sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Speed
If conventional nucleic acid detection methods are used, then detection accuracy is maintained, but detection speed and simplicity are insufficient for rapid diagnostics
Solution Approach 1:
The detection system is segmented into distinct functional modules: Cas12a/Cas12b proteins for target recognition, gRNA for sequence-specific binding, and fluorophore-quencher reporters for signal generation. This modular segmentation enables rapid parallel processing of multiple targets while maintaining operational simplicity through standardized component interfaces.
Solution Approach 2:
The patent replaces complex mechanical detection systems with biochemical reactions. The Cas12a/12b enzymes automatically recognize and cleave target sequences, and the fluorophore-quencher reporters autonomously generate signals upon cleavage, eliminating the need for complex mechanical manipulation and simplifying the detection process while accelerating results.
2Adaptability or versatility
If multiplex detection capability is added to detect multiple nucleic acid targets simultaneously, then diagnostic versatility improves, but system complexity increases
Solution Approach 1:
The Cas12a and Cas12b proteins serve as universal platforms that can recognize diverse nucleic acid targets through different gRNA molecules. The same basic detection mechanism (Cas protein + gRNA + fluorophore-quencher reporter) applies to all targets, enabling multiplex detection without requiring separate complex systems for each target, thus maintaining simplicity while achieving versatility.
Solution Approach 2:
The patent uses identical structural templates for multiple detection channels: each target is detected using a copy of the same Cas protein-gRNA-reporter system with only the gRNA sequence varied. This copying approach allows simultaneous detection of multiple targets through parallel reactions, increasing versatility while keeping system complexity manageable through repetition of proven functional modules.
3Measurement precision
If CRISPR-based detection system is implemented, then detection accuracy and specificity are improved, but reagent complexity and cost increase
Solution Approach 1:
The patent achieves high detection accuracy by precisely controlling key parameters: the gRNA sequence is designed to match specific target regions, the fluorophore and quencher are positioned at optimal distances on the reporter molecule, and the Cas12a/12b proteins are used at optimized concentrations. These parameter optimizations enable specific target recognition and accurate signal generation while managing reagent complexity through systematic parameter control rather than structural 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 efficient and accurate multiplex detection of nucleic acids, enhancing the speed, simplicity, and accuracy of nucleic acid diagnostics, particularly in pathogen and genetic disease identification.
Implementation Method 1
a CRISPR-associated (Cas) protein, a guide RNA (gRNA), and a single-stranded nucleic acid reporter, and the gRNA includes a region to bind to the Cas protein and a guide sequence to hybridize with a target sequence on the target nucleic acid; and detecting a detectable signal generated due to cleavage of the Cas protein on the single-stranded nucleic acid reporter
Implementation Method 2
a guide sequence to hybridize with a target sequence on the target nucleic acid
Data Source
AI summary
A method for multiplex nucleic acid detection based on clustered regularly interspaced short palindromic repeat (CRISPR), a system, and a kit for detecting a target nucleic acid based on CRISPR are provided. The detection method includes: adding any one, any two, any three, or four from the group consisting of a first nucleic acid detection composition, a second nucleic acid detection composition, a third nucleic acid detection composition, and a fourth nucleic acid detection composition to a reaction system with a target nucleic acid to achieve the multiplex detection of the target nucleic acid.