CRISPR-Cas13 Detection Using Plasmonic Waveguide Microarrays
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Solution Overview
Problem
Current nucleic acid tests for SARS-CoV-2, particularly qRT-PCR, face limitations due to high false negative rates caused by viral mutations, requiring multiple primer and probe sets and being sensitive to sequence variations, which complicates detection and increases the risk of false negatives.
Innovation Solution
A CRISPR-Cas13 system comprising Cas13a protein and crRNA, with guide RNAs targeting specific sequences of the SARS-CoV-2 spike protein gene, utilizing titanium nitride nanocubes and a plasmonic waveguide microarray for thermoplasmonic amplification and signal detection, enhancing sensitivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If qRT-PCR is used for SARS-CoV-2 detection, then detection sensitivity is achieved, but false negative rate increases due to viral mutations
Solution Approach 1:
The patent divides the detection system into multiple independent crRNA components (crRNA-1, crRNA-2, crRNA-3) that each target different regions of the SARS-CoV-2 genome. This segmentation allows the system to detect multiple viral variants simultaneously, reducing false negatives caused by mutations in any single region.
Solution Approach 2:
The CRISPR-Cas13 system is designed with universal applicability across different SARS-CoV-2 variants. The multi-target crRNA design enables a single detection platform to identify multiple viral strains, making the system adaptable to emerging variants without requiring complete redesign of the detection methodology.
2Reliability
If multiple primer and probe sets are used to address mutations, then false negative rate decreases, but detection complexity increases
Solution Approach 1:
The patent combines multiple detection functions into a single CRISPR-Cas13 platform. Instead of using separate PCR assays for different targets, the system integrates multiple crRNA-guided detection pathways that converge on a unified signal amplification and detection mechanism, simplifying the overall detection workflow while maintaining high reliability.
Solution Approach 2:
The Cas13 protein serves as an intermediary that processes multiple crRNA targets through a common mechanism. Rather than requiring separate detection systems for each target, the Cas13 enzyme mediates all detection events through its collateral cleavage activity, which amplifies signals from multiple targets through a unified pathway.
3Productivity
If qRT-PCR targets short sequences, then amplification efficiency is high, but sensitivity to mutation decreases
Solution Approach 1:
The patent transitions from traditional PCR amplification to CRISPR-based detection with isothermal amplification (RPA). This dimensional change in the detection approach allows for longer target sequences to be detected without compromising amplification efficiency, as the isothermal method maintains high productivity while enabling broader genomic target coverage that is more resistant to mutation.
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 CRISPR-Cas13 system achieves 8%-25% higher detection sensitivity than qRT-PCR, providing a simple, sensitive, and specific method for accurately identifying SARS-CoV-2 nucleic acid, reducing false negatives and improving diagnostic accuracy.
Implementation Method 1
CRISPR-Cas13 system comprising Cas13a protein and crRNA, with guide RNAs targeting specific sequences of the SARS-CoV-2 spike protein gene
Implementation Method 2
utilizing titanium nitride nanocubes and a plasmonic waveguide microarray for thermoplasmonic amplification and signal detection
Data Source
AI summary
Provided herein is a CRISPR-Cas13 system, a kit and a method for detecting SARS-COV-2. The CRISPR-Cas13a system includes a Cas13a protein and crRNA, or a complex formed by Cas13a protein and crRNA. The crRNA includes a first guide RNA and a second guide RNA, the first guide RNA and the second guide RNA having at least one sequence selected from SEQ ID NOs: 1 to 33, respectively.