CRISPR-Cas3 DNA Detection With Single-Stranded Probe Cleavage
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Solution Overview
Problem
The molecular mechanism of DNA cleavage by the CRISPR-Cas3 system is not well understood, limiting its application in detecting target DNAs, and existing methods lack the specificity and sensitivity to detect trace amounts of target DNA.
Innovation Solution
A method utilizing the CRISPR-Cas3 system and a single-stranded probe DNA to detect target DNA by monitoring the cleavage of the probe, which occurs only when the CRISPR-Cas3 system correctly recognizes and binds to the target DNA, enabling high specificity and sensitivity, even in the presence of trace amounts.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If the CRISPR-Cas3 system is used to detect target DNA, then the specificity of detection is improved, but the sensitivity to detect trace amounts of target DNA is insufficient
Solution Approach 1:
The detection system is segmented into multiple functional components: the CRISPR-Cas3 complex for specific target recognition, and a separate single-stranded probe DNA that serves as a signal reporter. This segmentation allows the target recognition function and the signal generation function to operate independently, enabling high specificity through the CRISPR complex while achieving high sensitivity through the probe's cleavage signal that can be detected even at trace concentrations
Solution Approach 2:
The single-stranded probe DNA acts as an intermediary between the CRISPR-Cas3 system and the detection system. When the CRISPR-Cas3 complex recognizes and binds to the target DNA, it indirectly causes cleavage of the probe DNA through random cleavage activity. This intermediary mechanism translates the specific molecular recognition event into a detectable signal, amplifying the detection sensitivity while preserving the specificity of the CRISPR system
2Adaptability or versatility
If the molecular mechanism of CRISPR-Cas3 DNA cleavage is not understood, then the system cannot be effectively applied for detection, but elucidating the mechanism increases system complexity
Solution Approach 1:
The invention extracts and utilizes only the essential functional characteristics of the CRISPR-Cas3 system needed for detection purposes: the ability to specifically recognize and bind target DNA sequences, and the associated random cleavage activity on single-stranded DNA. By taking out these specific functions and separating them from the complete complex molecular mechanism, the system becomes applicable for detection without requiring full understanding or replication of all molecular details
Solution Approach 2:
The CRISPR-Cas3 system's natural random cleavage activity on single-stranded DNA is utilized as a built-in signal generation mechanism. Instead of adding external complexity to generate detection signals, the invention leverages the system's inherent property where target binding automatically triggers probe cleavage and signal production. This self-service approach enables detection functionality while maintaining relative simplicity
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 method achieves high specificity and sensitivity in detecting target DNA, allowing for rapid and accurate identification of viruses and mutations in various samples, including body fluids and genetic material, with the ability to detect as few as 1 to 10 copies of the target DNA.
Implementation Method 1
when the CRISPR-Cas3 system recognizes and binds to a target DNA in a sample
Implementation Method 2
the CRISPR-Cas3 system cleaves single-stranded DNAs (ssDNAs) present therearound at random
Data Source
AI summary
It has been found that by mixing a single-stranded probe DNA whose cleavage can be detected in a reaction system containing a CRISPR-Cas3 system and a sample from which to detect a target DNA, it is possible to detect the target DNA in the sample by using, as indication, a signal generated by the cleavage of the single-stranded probe DNA.