CRISPR Diagnostic System with Aptamer Signal Switch
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Solution Overview
Problem
Current nucleic acid detection methods face challenges in achieving high sensitivity and specificity while being cost-effective and portable, particularly in point-of-care settings, due to trade-offs among sensitivity, specificity, simplicity, and speed.
Innovation Solution
A nucleic acid detection system utilizing a CRISPR system with a guide RNA and an RNA-aptamer comprising quadruplex with enzymatic activity, where the enzymatic activity produces a color signal that is inactivated upon binding to target molecules, indicating their presence, and optionally includes nucleic acid amplification reagents.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If qPCR approaches are used for nucleic acid detection, then sensitivity is improved, but cost and device complexity increase, limiting usability to laboratory settings
Solution Approach 1:
The patent replaces complex mechanical/qPCR instrumentation with a biochemical CRISPR-based detection system that uses guide RNA-programmed effector proteins to specifically bind and cleave target nucleic acids, producing detectable signals through enzymatic reactions rather than mechanical amplification and detection mechanisms
Solution Approach 2:
The patent introduces guide RNA as an intermediary molecule that programs effector proteins to specifically recognize and bind target nucleic acid sequences, enabling highly specific detection without requiring complex instrumentation. The guide RNA acts as a molecular mediator between the detection system and the target
2Ease of operation
If isothermal nucleic acid amplification with portable platforms is used, then portability and simplicity are improved, but detection sensitivity decreases
Solution Approach 1:
The patent changes the detection parameters by using CRISPR effectors with high affinity for target nucleic acids and optimizing the enzymatic reaction conditions to achieve attomolar sensitivity levels, thereby maintaining high sensitivity in portable, isothermal conditions without requiring complex instrumentation
3Measurement precision
If CRISPR effector systems are used for nucleic acid detection, then specificity is improved through guide RNA programming, but system complexity increases due to multiple components required
Solution Approach 1:
The patent achieves universality by designing a modular CRISPR detection platform where guide RNAs can be programmatically redesigned to detect different target sequences, and effector proteins serve multiple functions including target recognition, cleavage, and signal generation. This multi-functionality reduces the need for separate components for each detection target
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 system enables sensitive and specific detection of nucleic acids with attomolar sensitivity, differentiating targets from non-targets based on single base pair differences, and can be prepared in a freeze-dried format for convenient point-of-care applications, suitable for viral detection, bacterial strain typing, and disease-associated cell-free DNA detection.
Implementation Method 1
an nucleic acid-aptamer comprising quadruplex having enzymatic activity
Implementation Method 2
the enzymatic activity of the quadruplex produces a color signal in the sample
Implementation Method 3
one or more guide RNAs designed to bind to corresponding target molecules
Implementation Method 4
The RNA-targeting effector protein may comprise one or more HEPN domains
Implementation Method 5
nucleic acid amplification reagents
Data Source
Figure 1
Figure 2A~2F
Figure 3
AI summary
The embodiments disclosed herein utilized RNA targeting effectors to provide a robust CRISPR-based diagnostic with attomolar sensitivity. Embodiments disclosed herein can detect both DNA and RNA with comparable levels of sensitivity and can differentiate targets from non-targets based on single base pair differences, and includes detection by colorimetric and/or fluorescence shifts. Moreover, the embodiments disclosed herein can be prepared in freeze-dried format for convenient distribution and point-of-care (POC) applications. Such embodiments are useful in multiple scenarios in human health including, for example, viral detection, bacterial strain typing, sensitive genotyping, and detection of disease-associated cell free DNA.