CRISPR SARS-CoV-2 Detection via Cas12a LAMP Integration
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Solution Overview
Problem
Current detection technologies for SARS-CoV-2 lack sensitivity and specificity, particularly in biological samples, and require multiple processing steps and equipment, making them inefficient for rapid and accurate diagnosis.
Innovation Solution
The use of CRISPR/Cas enzymes, specifically thermostable Cas12 or Cas13 enzymes, in combination with loop-mediated isothermal amplification (LAMP) and labeled nucleic acid reporter constructs, for the detection of SARS-CoV-2 nucleic acids in a single reaction vessel, enabling sensitive and specific detection without the need for extensive sample processing.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional detection technologies are used for SARS-CoV-2, then detection can be performed, but sensitivity and specificity are insufficient particularly in biological samples
Solution Approach 1:
The patent combines CRISPR/Cas12a or Cas13b enzymes with LAMP amplification and fluorescent reporter constructs in a single reaction system. This integration allows the CRISPR enzyme to amplify target nucleic acids while simultaneously providing specific detection through fluorescent signal generation, achieving high sensitivity and specificity for SARS-CoV-2 detection in biological samples
Solution Approach 2:
The patent introduces fluorescent reporter constructs as intermediaries between the CRISPR/Cas enzymes and the detection signal. These reporters are cleaved or activated by the CRISPR enzymes upon binding to the target, converting the molecular interaction into a measurable fluorescent signal that enables sensitive and specific detection
2Productivity
If conventional detection methods are used, then SARS-CoV-2 can be detected, but multiple processing steps and equipment are required making them inefficient for rapid diagnosis
Solution Approach 1:
The patent merges amplification, detection, and signal generation into a single reaction vessel that can be performed isothermally. This eliminates the need for multiple separate processing steps and complex equipment, enabling rapid diagnosis while maintaining high sensitivity and specificity for SARS-CoV-2 detection
Solution Approach 2:
The CRISPR/Cas12a or Cas13b enzymes perform multiple functions simultaneously: they amplify the target nucleic acid sequence and generate the detection signal through collateral cleavage of fluorescent reporters. This self-service capability eliminates the need for separate amplification and detection systems, simplifying the overall process for rapid diagnosis
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 significantly enhances the sensitivity and specificity of SARS-CoV-2 detection, allowing for the identification of as few as 3-53 viral copies per microliter and reducing processing complexity, thereby improving diagnostic efficiency and accuracy.
Implementation Method 1
CRISPR/Cas enzymes, specifically thermostable Cas12 or Cas13 enzymes, in combination with loop-mediated isothermal amplification (LAMP) and labeled nucleic acid reporter constructs, for the detection of SARS-CoV-2 nucleic acids
Implementation Method 2
loop-mediated isothermal amplification (LAMP) and labeled nucleic acid reporter constructs, for the detection of SARS-CoV-2 nucleic acids
Implementation Method 3
labeled nucleic acid reporter constructs, for the detection of SARS-CoV-2 nucleic acids in a single reaction vessel
Data Source
AI summary
The present disclosure provides methods and compositions for the detection of SARS-CoV-2 by CRISPR/Cas collateral RNAse activity.