Photo-Controlled CRISPR-Cas Detection Kit for Closed-System Nucleic Acid Analysis
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Solution Overview
Problem
Current CRISPR-Cas nucleic acid detection methods face challenges in sensitivity due to the need for nucleic acid amplification and step-by-step processing, which leads to aerosol pollution and reduced efficiency, necessitating a method that enhances sensitivity while avoiding aerosol contamination.
Innovation Solution
A photo-controlled CRISPR-Cas system that separates nucleic acid amplification from detection, using a silent guide RNA and Cas protein complex with a photo-activated linker, allowing for isothermal amplification followed by UV activation for CRISPR-Cas cleavage in a closed tube, preventing aerosol pollution and maintaining high sensitivity.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Device complexity
If nucleic acid amplification and CRISPR-Cas detection are combined in a single reaction tube, then the detection process is simplified and closed, but the CRISPR-Cas system will recognize and cleave amplification templates, reducing amplification efficiency
Solution Approach 1:
The patent divides the detection process into two distinct phases: a nucleic acid amplification phase and a CRISPR-Cas detection phase. The silent guide RNA is designed to be inactive during amplification and only becomes active after UV irradiation, effectively segmenting the functions to prevent interference between amplification and detection processes.
Solution Approach 2:
The silent guide RNA is designed with dynamic properties - it transitions from an inactive state during amplification to an active state after UV irradiation. This dynamic activation allows the same component to serve different functions at different stages without causing interference.
2Productivity
If step-by-step processing is used for nucleic acid amplification and CRISPR-Cas detection, then amplification efficiency is maintained, but aerosol pollution occurs during tube uncapping and liquid transfer
Solution Approach 1:
The patent merges both amplification and detection processes into a single closed tube, eliminating the need for tube uncapping and liquid transfer between steps. The silent guide RNA enables the CRISPR-Cas system to function within the same closed reaction tube after UV activation, maintaining a closed-system approach throughout.
Solution Approach 2:
The detection process continues within the same closed tube without interruption or transfer. After amplification, the silent guide RNA is activated by UV irradiation and immediately begins detecting target nucleic acids in the same tube, maintaining continuous operation without opening the system.
3Measurement precision
If CRISPR-Cas detection is performed before amplification, then detection sensitivity is improved, but the initial amplification products will be recognized and cleaved, breaking templates that can be recycled
Solution Approach 1:
The silent guide RNA is prepared in advance in an inactive state, allowing amplification to proceed first without interference. The UV irradiation activation step is performed preliminarily before detection begins, ensuring that the CRISPR-Cas system is activated only after amplification is complete, preventing premature cleavage of amplification products.
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 approach simplifies the detection process, avoids aerosol pollution, and achieves high sensitivity by enabling CRISPR-Cas detection in a closed system without affecting nucleic acid amplification, improving experimental efficiency and stability.
Implementation Method 1
bases of the silent nucleotide are linked by PC linker that can be photo-activated and photo-degraded
Data Source
AI summary
The present invention discloses a nucleic acid detection kit and a detection method based on photo-controlled CRISPR-Cas, wherein the kit comprises silent guide RNA and Cas protein; the silent guide RNA is formed by annealing hybridization of silent nucleotide and guide RNA; the guide RNA, designed according to a target nucleic acid sequence, includes two regions, i.e. a repetitive region and a spacer region; the silent nucleotide is completely complementarily paired with a the of the guide RNA, or is completely paired with a the of the guide RNA; the bases of the silent nucleotide are linked by PC linker; and the Cas protein is Cas12 protein or Cas13 protein. Although this method separates the nucleic acid amplification from the CRISPR-Cas detection in time, it can allow them to be completed in the same closed reaction tube, thereby avoiding the transfer process of uncapped reagent, ensuring that the detection is not affected by aerosol pollution while ensuring high detection sensitivity.