dCas9/gRNA Complex Target RNA Detection Without Amplification
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Solution Overview
Problem
Current methods for detecting target RNA are limited by low sensitivity and specificity, especially when dealing with small amounts of nucleic acid, and require separate gene isolation and amplification steps, making them inefficient for rapid diagnosis of viral infections.
Innovation Solution
A target RNA detection method using a dCas9/gRNA complex with a PAMmer, which includes a labeled ligand and specific hybridization regions, allowing for direct detection without amplification, enabling high sensitivity and specificity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional PCR-based detection methods are used, then amplification capability is improved, but detection time and process complexity increase
Solution Approach 1:
The invention extracts and eliminates the unnecessary PCR amplification step from the detection process. By using a detection system that directly binds to target RNA through CRISPR-Cas9/gRNA complex formation, the method removes the time-consuming gene isolation and amplification steps while maintaining detection capability through direct molecular recognition.
Solution Approach 2:
The invention performs preliminary preparation of the CRISPR-Cas9/gRNA complex before detection, allowing the complex to be pre-formed and ready for immediate binding to target RNA. This preliminary action eliminates the need for real-time amplification during detection, significantly reducing detection time while maintaining sensitivity.
2Measurement precision
If gene isolation and amplification steps are performed, then detection sensitivity is improved, but process complexity increases
Solution Approach 1:
The invention extracts and removes the complex gene isolation and amplification steps from the detection workflow. By utilizing the high specificity of CRISPR-Cas9/gRNA binding to target RNA sequences, the method achieves detection sensitivity without requiring multiple processing steps, thereby simplifying the overall process.
Solution Approach 2:
The invention introduces the CRISPR-Cas9/gRNA complex as an intermediary that directly binds to target RNA. This intermediary mechanism provides high detection sensitivity through specific molecular recognition without requiring the complex amplification processes used in conventional methods.
3Measurement precision
If labeled monomers are used in PCR, then labeling capability is improved, but PCR efficiency decreases
Solution Approach 1:
The invention extracts and eliminates the PCR amplification process entirely, removing the trade-off between labeling capability and PCR efficiency. By using direct CRISPR-Cas9/gRNA binding with labeled probes, the method achieves labeling capability without the efficiency loss associated with labeled monomer incorporation during PCR.
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
Enables rapid and accurate detection of target RNA with high sensitivity and specificity, allowing for the identification of pathogens and viruses, including those with single nucleotide mutations, without the need for separate gene isolation and amplification steps.
Implementation Method 1
a gRNA (guide RNA) complementary to a target RNA
Implementation Method 2
a labeled ligand indirectly generating a detectable signal is bound to 3'-end
Data Source
AI summary
The present invention provides a target RNA detection method based on a dCas9/gRNA complex. A target RNA detection method according to the present invention can detect target RNA with the naked eye and without separate gene isolation and amplification steps, and, in particular, can rapidly and accurately detect target RNA through excellent target specificity and rapidity, and thus can exhibit excellent effects on the detection of various pathogens and/or viruses.