CRISPR RNA Detection with Catcher Polynucleotides
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Solution Overview
Problem
Current RNA detection methods using CRISPR/Cas systems are limited in their diagnostic capabilities and do not fully exploit the potential of RNA-based CRISPR/Cas systems, necessitating new approaches for efficient and accurate characterization of RNA polynucleotides.
Innovation Solution
A method involving catcher polynucleotides complementary to target RNA, a mature crRNA molecule specific to the target sequence, and a catalytically-inactive crRNA-guided RNA-binding protein, with subsequent antibody binding and detection, allowing for improved dynamic range, adaptability, and high specificity in RNA detection.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If CRISPR/Cas systems are used for RNA detection, then specificity is improved, but diagnostic capability and adaptability are limited
Solution Approach 1:
The detection system is segmented into distinct functional modules: catcher polynucleotides for target capture, crRNA for sequence-specific recognition, and catalytically-inactive Cas proteins for signal generation. This modular architecture enables independent optimization of each component and facilitates adaptation to different diagnostic applications by simply changing the crRNA sequence while maintaining the core detection machinery.
Solution Approach 2:
The patent creates a universal detection platform that can detect various RNA targets by using the same core components (catcher polynucleotides, crRNA, and Cas protein) with only the crRNA sequence needing to be changed. This multi-functional system maintains high specificity through CRISPR-based recognition while achieving broad adaptability across different diagnostic scenarios including infectious diseases, genetic disorders, and cancer detection.
2Measurement precision
If complex detection protocols are used, then detection accuracy is improved, but procedure time is increased
Solution Approach 1:
The catcher polynucleotides are pre-immobilized on solid supports and the Cas proteins are pre-prepared in catalytically-inactive forms ready for immediate use. The crRNA is designed with optimal sequences that require minimal processing. These preliminary preparations eliminate time-consuming setup steps during actual detection, allowing rapid execution of the assay while maintaining high detection accuracy through the pre-optimized components.
Solution Approach 2:
The patent employs a streamlined protocol that skips unnecessary intermediate steps found in traditional methods. The catalytically-inactive Cas proteins are directly activated upon binding to the target RNA-crRNA complex, eliminating separate activation steps. The assay proceeds through rapid sequential binding events that can be completed in a single incubation step, significantly reducing total procedure time while preserving detection accuracy.
3Ease of operation
If manual detection methods are used, then flexibility is maintained, but automation compatibility is reduced
Solution Approach 1:
The patent replaces manual mechanical operations with biochemical and optical detection systems. The catcher polynucleotides immobilized on solid supports enable automated washing and binding steps. The catalytic activation of Cas proteins and subsequent detection of reaction products can be performed using automated plate readers or other high-throughput instrumentation, seamlessly integrating manual flexibility with automated efficiency.
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 efficient and accurate detection of RNA molecules with improved dynamic range, adaptability to various sequences, and suitability for automated platforms, providing high specificity and short procedure times, making it suitable for both laboratory and point-of-care settings.
Implementation Method 1
annealing one or more catcher polynucleotides which are complementary to at least a portion of the target RNA polynucleotide
Implementation Method 2
catcher polynucleotides which are complementary to at least a portion of the target RNA polynucleotide
Implementation Method 3
annealing a mature crRNA (CRISPR RNA) molecule to a specific target sequence on the target RNA polynucleotide
Implementation Method 4
mature crRNA (CRISPR RNA) molecule which is specific for a target sequence on the target RNA polynucleotide
Implementation Method 5
binding a catalytically-inactive crRNA-guided RNA-binding protein to the complex of step (iii) to obtain a protein-RNA polynucleotide complex
Implementation Method 6
binding of an antibody to said protein-RNA polynucleotide-complex of step (iv)
Data Source
Figure 1
AI summary
The present invention relates to a method for detecting a target RNA polynucleotide by using a catcher polynucleotide which anneals to at least a portion of the target RNA polynucleotide, and a catalytically-inactive crRNA-guided (CRISPR RNA) RNA-binding protein which binds to the target RNA polynucleotide. Further envisaged is a kit for detecting a specific target RNA polynucleotide comprising inter alia one or more catcher polynucleotides complementary to at least a portion of the target RNA polynucleotide, a mature crRNA molecule which is specific for a target sequence on the target RNA polynucleotide and a catalytically-inactive crRNA-guided RNA-binding protein; as well as the use of these ingredients for the detection of the target RNA polynucleotide.