Guide RNA Cas Complex Immobilized Solid Support Nucleic Acid Detection
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Solution Overview
Problem
Current nucleic acid detection methods struggle with sensitivity and reliability, especially at low concentrations, and lack the capability for multiplex assays to detect multiple targets simultaneously.
Innovation Solution
The use of guide RNA (gRNA) and gRNA/Cas complexes immobilized on a solid support allows for the detection of target nucleic acids, even at very low concentrations, by deactivating the Cas enzyme's cleavage activity and enhancing capture efficiency.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If quantitative polymerase chain reaction is used for nucleic acid detection, then sensitivity and specificity are improved, but device complexity and cost increase
Solution Approach 1:
The patent extracts the essential detection function from complex PCR systems by using simplified gRNA/Cas complex binding assays. The method removes amplification steps and complex instrumentation, retaining only the core nucleic acid recognition capability through guide RNA-directed Cas enzyme binding to target sequences, enabling detection without sophisticated thermal cycling equipment
Solution Approach 2:
The patent uses guide RNA molecules as informational copies that direct the Cas enzyme to specific target nucleic acid sequences. The gRNA serves as a simplified informational template that replicates the specificity function of complex PCR primers and probes, enabling target recognition through sequence complementarity rather than amplification
2Productivity
If Cas enzyme cleavage activity is activated for target detection, then target capture efficiency is improved, but target nucleic acid integrity is compromised
Solution Approach 1:
The patent applies preliminary anti-action by deactivating the Cas enzyme's cleavage activity before the detection assay. Mutations are introduced into the Cas enzyme to prevent cleavage of target nucleic acids, ensuring target integrity is preserved while the enzyme retains its ability to bind to target sequences through gRNA-directed recognition
Solution Approach 2:
The patent converts the potentially harmful cleavage activity into a beneficial binding function. By disabling the cleavage capability, the Cas enzyme becomes a stable, specific binder that can be used for detection without destroying the target, while still maintaining the high affinity and specificity originally provided by the cleavage mechanism
3Ease of operation
If single target detection methods are used, then assay simplicity is maintained, but multiplexing capability is lost
Solution Approach 1:
The patent segments the detection function into modular gRNA/Cas complex units, where each gRNA is designed to recognize a specific target sequence. Multiple different gRNA/Cas complexes can be used in the same assay, with each targeting a different nucleic acid sequence, enabling simultaneous detection of multiple targets while maintaining the simplicity of individual binding assays
Solution Approach 2:
The patent creates a universal detection platform where the Cas enzyme serves as a common binding platform that can recognize multiple different target sequences through exchangeable gRNA molecules. This multi-functional system allows a single Cas enzyme type to detect various targets by simply changing the guide RNA, enabling multiplexing without requiring multiple different enzymatic systems
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 enables the sensitive and specific detection of multiple target nucleic acids in a single assay, improving the accuracy and efficiency of nucleic acid detection.
Implementation Method 1
the contacting is under conditions in which the target nucleic acid hybridizes to the complementary gRNA molecule on the apparatus
Data Source
AI summary
Methods, microarrays, and kits for detecting target nucleic acid molecules (e.g., in a sample) using immobilized guide RNA (gRNA), or gRNA/Cas complexes, are provided herein. In some embodiments disclosed herein, a plurality of guide RNA (gRNA) molecules are bound, directly or indirectly, to a solid support, wherein at least one gRNA molecule in the plurality comprises a nucleotide sequence that is complementary to a portion of the target nucleic acid in the sample.