Split-Luciferase Reporter Systems for Multiplex CRISPR Diagnostics
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Solution Overview
Problem
Current diagnostic technologies for infectious diseases lack sensitivity, specificity, deployability, speed, and multiplexing capabilities required for a broad and robust response to outbreaks.
Innovation Solution
Development of a split-luciferase reporter system coupled with a CRISPR-Cas system, utilizing color-coded bead multiplexing for enhanced sensitivity, deployability, and multiplexing in diagnostic assays.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If qPCR is used for pathogen detection, then sensitivity and specificity are improved, but deployability and multiplexing capability deteriorate
Solution Approach 1:
The luciferase reporter protein is divided into two separate subunits (first and second subunits) that are attached to different beads. These segmented subunits only assemble to produce light when brought together by CRISPR-Cas cleavage, enabling sensitive detection while simplifying the overall system architecture for better deployability
Solution Approach 2:
The CRISPR-Cas system serves multiple functions: it provides target-specific recognition through guide RNA, performs collateral cleavage to release the reporter subunits, and enables multiplexing through color-coded beads. This multi-functional approach replaces the need for separate detection systems for each pathogen, improving both deployability and multiplexing capability
2Ease of operation
If fluorescence-based CRISPR-Cas systems are used, then deployability is improved, but sensitivity deteriorates
Solution Approach 1:
The system replaces fluorescence-based detection with a luminescence-based reporter system. Luminescence provides inherently higher sensitivity due to its lower background signal, while maintaining the portability and ease of operation of CRISPR-Cas systems. The chemical luminescence reaction occurs without requiring complex optical equipment
Solution Approach 2:
The patent changes the detection parameter from fluorescence emission to luminescence intensity. This parameter change fundamentally improves sensitivity because luminescence has no background signal (unlike fluorescence which must overcome autofluorescence), thereby achieving high sensitivity while maintaining deployability
3Adaptability or versatility
If multiplexed pathogen detection is implemented, then adaptability is improved, but device complexity increases
Solution Approach 1:
The patent adds a color dimension to the detection system by using color-coded beads. Each bead color represents a different pathogen target, allowing multiplexed detection in a single assay. This dimensional approach (adding color coding) enables complex multiplexing functionality without proportionally increasing operational complexity
Solution Approach 2:
Multiple pathogen detection capabilities are merged into a single reaction well by combining color-coded beads with different CRISPR-Cas targets in one assay. This merging approach allows simultaneous detection of multiple pathogens without requiring separate tests, thereby improving adaptability while managing system complexity through integration
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
The system achieves improved sensitivity and multiplexing capabilities, enabling rapid and deployable detection of multiple pathogens in a single assay, addressing the limitations of existing technologies.
Implementation Method 1
the first and second reporter protein subunits can complex to generate a detectable luminescent signal
Implementation Method 2
coupled to first bead or a masking agent via a first oligonucleotide linker capable of being cleaved by Cas nuclease activity
Data Source
AI summary
Cas-cleavable reporter systems, CRISPR-Cas systems thereof, and methods of use thereof in CRISPR-Cas based diagnostics. Cas-cleavable reporter systems may generate a luminescent, fluorescent, or other detectable signal upon Cas-collateral cleavage of one or more reporter system components. CRISPR-Cas systems may comprise a Cas protein having collateral cleavage activity, guide molecules, and the Cas-cleavable reporter system. Cas-cleavable reporter systems may be a split luciferase reporter system, at least one element of which is bound to beads. For multiplexing, CRISPR-Cas systems may comprise a Cas-cleavable quenched reporter system, optionally a Cas-cleavable quenched fluorescent reporter system, a Cas protein having collateral cleavage activity, and target-specific guide molecules attached to color-coded beads. CRISPR-Cas systems may further comprise amplification reagents. Methods may apply said CRISPR-Cas systems to flow cell, well-plate, or other devices, and may measure detectable signals by microscopic, plate reader, or other methods.