CRISPR/Cas Collateral Nuclease Signal Amplification
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Solution Overview
Problem
Current methods for detecting nucleic acids, such as PCR and isothermal nucleic acid amplification, lack sensitivity, specificity, and are costly, making them unsuitable for rapid, cost-effective, and versatile point-of-care applications.
Innovation Solution
The use of CRISPR/Cas complexes with collateral nuclease activity, which includes a guide RNA, effector nuclease, and additional nucleases and oligonucleotides, to amplify a fluorescence signal for detecting target nucleic acids without pre-amplification, enabling high sensitivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If PCR is used for detecting nucleic acids, then detection sensitivity and specificity are improved, but device complexity and cost increase
Solution Approach 1:
The patent replaces the mechanical/thermal cycling system of PCR with a biochemical CRISPR-based detection system. The CRISPR/Cas complex performs sequence-specific recognition and cleavage at constant temperature, eliminating the need for thermocyclers and complex temperature cycling equipment while maintaining high detection sensitivity and specificity.
Solution Approach 2:
The patent introduces reporter molecules (fluorogenic substrates) as intermediaries that amplify the detection signal. When the CRISPR/Cas complex cleaves the target nucleic acid, it activates collateral nuclease activity that cleaves reporter molecules, generating a measurable fluorescence signal that indirectly indicates target presence, thereby enhancing sensitivity without requiring complex instrumentation.
2Speed
If isothermal nucleic acid amplification is used, then speed is improved, but detection sensitivity and specificity deteriorate
Solution Approach 1:
The patent segments the detection function into two distinct parts: (1) the CRISPR/Cas complex provides high-specificity sequence recognition and discrimination through guide RNA-target pairing, and (2) the collateral nuclease activity provides rapid signal amplification. This segmentation allows the system to achieve both high speed and high specificity simultaneously, overcoming the limitation of isothermal amplification methods.
3Measurement precision
If pre-amplification steps are added to improve sensitivity, then detection sensitivity is improved, but procedure complexity and time increase
Solution Approach 1:
The patent employs a self-amplifying CRISPR/Cas system where the activated complex automatically generates collateral nuclease activity that cleaves reporter molecules to amplify the detection signal. This self-service mechanism provides signal amplification without requiring external pre-amplification steps, reducing procedural complexity and time while maintaining high detection sensitivity.
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 allows for the rapid detection of nucleic acids with attomolar sensitivity and specificity, distinguishing similar sequences, and can be used for various nucleic acid types, including viral, bacterial, and parasitic nucleic acids, without the need for complex equipment or pre-amplification.
Implementation Method 1
a guide RNA that encodes a nucleic acid complementary to a target sequence
Implementation Method 2
measuring a fluorescence signal emitted from the fluorescence reporter
Implementation Method 3
Cas effector nucleases from particular types of CRISPR/Cas complexes have been found to exhibit target-dependent promiscuous nuclease RNase activity (e.g., RNase activity and DNase activity), leading to trans cleavage of bystander RNA/DNA molecules
Data Source
AI summary
Provided herein are methods that utilize a CRISPR/Cas complex having collateral activity, one or more nucleases, one or more oligonucleotides and a fluorescent reporter. The methods disclosed herein can amplify a fluorescent signal when a target nucleic acid is present in a sample.