CrisprZyme Nanozyme Detection of Nucleic Acids
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Solution Overview
Problem
Current CRISPR-based nucleic acid detection systems require preamplification steps like PCR or isothermal amplification, which add complexity, limit quantification capability, and are not suitable for point-of-care diagnostics due to thermal requirements and lack specificity for short targets like microRNAs.
Innovation Solution
The CrisprZyme system combines a CRISPR-based reaction with a Nanozyme-Linked ImmunoSorbent Assay (NLISA) using catalytic nanoparticles to quantify cleaved reporter RNA without preamplification, offering high sensitivity and ease of use.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If preamplification steps (PCR or isothermal amplification) are used to increase sensitivity, then detection sensitivity is improved, but device complexity and operational requirements increase
Solution Approach 1:
The invention extracts and eliminates the preamplification step from the CRISPR diagnostic workflow. By using a signal amplification strategy instead of target amplification, the complex PCR or isothermal amplification machinery is removed, leaving only the essential Cas13a recognition and cleavage functions, thereby simplifying the overall reaction chemistry while maintaining detection sensitivity
Solution Approach 2:
The invention changes the amplification parameter from target DNA/RNA copying to signal molecule multiplication. Instead of increasing the amount of target through exponential copying (PCR/isothermal), the system uses Cas13a-mediated cleavage of numerous reporter RNA molecules to generate amplified signals, fundamentally altering how sensitivity is achieved without requiring complex amplification devices
2Measurement precision
If preamplification steps are used to reach clinically relevant concentrations, then detection sensitivity is improved, but quantification capability is limited
Solution Approach 1:
The invention segments the detection process into distinct phases: specific target recognition by Cas13a-gRNA complex, followed by catalytic cleavage of reporter RNA. This segmentation allows the quantification to be performed on the linear relationship between target molecules and cleaved reporters, avoiding the exponential non-linearity introduced by preamplification steps and thereby preserving quantification accuracy
3Measurement precision
If PCR thermal cycling is used for amplification, then detection sensitivity is improved, but ease of operation is reduced due to thermal requirements
Solution Approach 1:
The invention substitutes the mechanical thermal cycling system with a constant-temperature biochemical system. Cas13a-mediated cleavage operates efficiently at constant physiological temperatures (37°C or lower), replacing the complex thermal cycling machinery of PCR with simple isothermal conditions that can be maintained in basic water baths or even body temperature, dramatically improving ease of operation for point-of-care settings
4Ease of operation
If isothermal amplification is used to avoid thermal cycling, then ease of operation is improved, but specificity for short targets like microRNAs is reduced
Solution Approach 1:
The invention performs preliminary specific recognition through the Cas13a-gRNA complex formation before any signal generation occurs. The gRNA is designed with perfect complementarity to the target microRNA sequence, ensuring high specificity is established in advance through molecular recognition, and this specific complex then triggers the subsequent isothermal cleavage reaction, combining specificity with operational 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
CrisprZyme achieves a 1000-fold improvement in sensitivity, enabling the detection of non-coding RNAs, including microRNAs, and allows for quantitative, preamplification-free diagnostics in complex samples and resource-limited settings.
Implementation Method 1
sensing of DNA or RNA is mediated through a complementary guide RNA (gRNA), which induces the activation of a Cas enzyme that indicates the presence of a target analyte
Implementation Method 2
NLISA quantifies the cleaved reporter RNA by catalysing the readout signal; it is a stepwise addition of reagents onto an immobilised surface that ends with the catalysis of a chromogenic substrate to generate a readout signal
Data Source
AI summary
The present disclosure relates to systems and methods of detecting nucleic acids using a combination of a Cas-based reaction with catalytic nanoparticles. Provided is a nucleic acid detection system for detecting one or more target nucleic acids, the system comprising a CRISPR effector protein; one or more guide RNAs (gRNA), each of which is specific for the one or more target nucleic acids; and a reporter RNA molecule; and a catalytic nanoparticle. Also provided is a reporter RNA molecule for a CRISPR detection assay. The disclosure also relates to uses, methods and kits for detecting one or more target nucleic acids. The uses, methods and kits extend to diagnosing a disease in a patient based on the presence of a nucleic acid of interest.