Multiplex CRISPR Assay for Chlamydia and Gonorrhea Detection
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Solution Overview
Problem
Current diagnostic methods for Chlamydia trachomatis and Neisseria gonorrhoeae infections are inadequate due to their lack of accuracy, high costs, and requirement for complex equipment, making them unsuitable for rapid and effective detection, especially in resource-limited settings.
Innovation Solution
A CRISPR-directed nucleic acid detection assay using CRISPR nuclease with collateral nuclease activity and crRNA specific to Chlamydia trachomatis and Neisseria gonorrhoeae, which cleaves single-stranded linker probes in the presence of target DNA, allowing for rapid and accurate detection using lateral flow assays without the need for additional amplification steps.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If current diagnostic methods are used for Chlamydia and Neisseria detection, then detection accuracy can be maintained, but the cost increases and equipment complexity increases
Solution Approach 1:
The patent replaces complex mechanical/electronic diagnostic equipment with a biochemical CRISPR-based detection system. The Cas12a nuclease complex with crRNA performs specific DNA recognition and collateral cleavage of reporter molecules, eliminating the need for sophisticated instrumentation while maintaining high detection accuracy through sequence-specific molecular recognition
Solution Approach 2:
The CRISPR-Cas12a system performs self-amplification and self-detection functions. The collateral nuclease activity automatically amplifies the detection signal through indiscriminate cleavage of single-stranded DNA reporters, eliminating the need for external amplification equipment or complex mechanical systems
2Measurement precision
If current diagnostic methods are used for Chlamydia and Neisseria detection, then detection accuracy can be maintained, but the cost increases
Solution Approach 1:
The patent uses inexpensive, easily synthesized crRNA molecules and Cas12a protein as disposable reagents. These biochemical components can be produced through simple transcription and purification processes, replacing expensive proprietary diagnostic kits and specialized equipment while maintaining equivalent or superior detection accuracy
Solution Approach 2:
The patent changes the detection parameters from instrument-dependent measurements to biochemical reaction outcomes. By measuring collateral cleavage activity of Cas12a on reporter DNA, the system achieves high accuracy through molecular recognition parameters rather than expensive instrumental measurements
3Loss of time
If rapid detection is implemented, then time is reduced, but accuracy may be compromised
Solution Approach 1:
The patent performs preliminary target recognition and binding through the crRNA-Cas12a complex formation before detection. The specific binding of crRNA to complementary DNA sequences occurs rapidly at room temperature, establishing accurate target identification before the collateral cleavage amplification step, thus ensuring both speed and accuracy
Solution Approach 2:
The CRISPR-Cas12a system maintains continuous useful action through collateral nuclease activity. Once the complex binds to target DNA, it continuously cleaves reporter molecules, generating a sustained amplification signal that rapidly increases detection sensitivity without compromising accuracy, allowing results in minutes rather than hours
4Ease of operation
If simple detection methods are used, then ease of operation is improved, but accuracy deteriorates
Solution Approach 1:
The patent introduces crRNA as an intermediary molecule that mediates specific recognition between Cas12a and target DNA. This RNA mediator provides high specificity through sequence complementarity while allowing the system to operate simply through mixing reagents and incubation, eliminating complex equipment while maintaining accuracy through molecular mediation
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 method provides a fast, cost-effective, and highly accurate diagnostic tool that can be performed at the point of care, reducing overtreatment and undertreatment, and minimizing the risk of antibiotic resistance, while enabling timely infection control.
Implementation Method 1
CRISPR nuclease with collateral nuclease activity and crRNA specific to Chlamydia trachomatis and Neisseria gonorrhoeae, which cleaves single-stranded linker probes in the presence of target DNA
Implementation Method 2
crRNA comprising a nucleic acid sequence that is complementary to a nucleic acid sequence of Chlamydia trachomatis and Neisseria gonorrhoeae
Data Source
AI summary
The present invention provides methods of detecting the presence or absence of nucleic acid from Chlamydia trachomatis and the presence or absence of nucleic acid from Neisseria gonorrhoeae in a sample from a subject using CRISPR nucleases and crRNAs specific for Chlamydia trachomatis and Neisseria gonorrhoeae nucleic acid, kits for carrying out the methods and methods of treating subjects infected with Chlamydia trachomatis and/or Neisseria gonorrhoeae.