High-Throughput CRISPR Diagnostics with Spatially Segregated Guide Molecules
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Solution Overview
Problem
Current diagnostic methods lack the ability to rapidly detect nucleic acids with high sensitivity and single-base specificity for a large number of samples in a timely manner, limiting their effectiveness in disease diagnosis and monitoring.
Innovation Solution
A high-throughput method utilizing spatially segregated CRISPR guide molecules, distributed in individual discrete volumes, that form complexes with Cas proteins to cleave reporter constructs and generate detectable signals, enabling simultaneous detection of multiple targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If current diagnostic methods are used, then detection can be performed, but the ability to rapidly detect nucleic acids with high sensitivity and single-base specificity for a large number of samples is limited
Solution Approach 1:
The diagnostic system divides the detection process into spatially segregated discrete volumes, each containing specific guide molecules targeted to particular nucleic acid sequences. This segmentation enables parallel processing of multiple samples simultaneously while maintaining high detection precision through isolated reaction compartments.
Solution Approach 2:
The patent introduces guide molecules as intermediary elements that bridge the Cas protein and target nucleic acids. These guide molecules facilitate specific binding and activation of Cas proteins against target sequences, enabling sensitive and specific detection without direct Cas protein-target interaction.
2Reliability
If guide molecules are distributed in individual discrete volumes, then spatial segregation is achieved, but the complexity of distribution and volume management increases
Solution Approach 1:
Each discrete volume is designed with specific local qualities - different guide molecules, concentrations, and configurations tailored to detect particular targets. This local differentiation enables reliable spatial segregation while using standardized volume formats that simplify distribution logistics.
Solution Approach 2:
The system varies key parameters such as guide molecule sequence, concentration, and volume size to create distinguishable detection channels. These parameter changes enable reliable segregation of detection functions while maintaining compatibility with standard microplate formats, reducing overall system complexity.
3Adaptability or versatility
If multiple sets of guide molecules are distributed to detect multiple target molecules, then detection capability expands, but the amount of reagents and sample consumption increases
Solution Approach 1:
Multiple guide molecules and detection components are nested within individual discrete volumes, with each volume containing a complete detection system for specific targets. This nesting enables high versatility for detecting multiple targets while minimizing reagent consumption through efficient use of contained volumes.
Solution Approach 2:
The system uses partial volumes optimized for high-throughput screening, where each discrete volume contains sufficient reagents for the intended detection task without excess. This approach enables expanded multi-target detection capability while controlling overall reagent and sample consumption through precise volume management.
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, sensitive, and specific detection of nucleic acids at nanoliter volumes, differentiating targets from non-targets based on single base pair differences, facilitating efficient disease diagnosis and monitoring.
Implementation Method 1
guide molecules capable of binding one or more target sequences of a target molecule
Implementation Method 2
the Cas protein cleaves the non-target sequence of the reporter constructs once activated by the target sequences
Data Source
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Figure 4~5A
Figure 5B~6A
AI summary
High throughput methods utilizing spatially segregated detection systems provide a robust CRISPR-based diagnostic enabling highly sensitive detection of both DNA and RNA target molecules, with applications in multiple scenarios in human health including, for example, viral detection. Kits comprising the systems allow for point-of care applications with high-throughput processing of samples.