Split Intein Reporter for Splice Event Detection
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Solution Overview
Problem
Current methods for detecting specific splice events in genes lack high-throughput and spatiotemporal resolution, making it difficult to track and diagnose neurological and neuromuscular diseases such as tauopathies, which are associated with imbalances in tau protein isoforms, and are not suitable for real-time monitoring or early-stage disease detection.
Innovation Solution
A method involving the insertion of a split intein-heterologous polynucleotide construct into an exon of interest, where the split intein excises itself from the expression product, allowing for the detection of specific splice events with high-throughput and spatiotemporal resolution, and includes the use of CRISPR/Cas9 for precise gene editing and monitoring.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If laborious methods based on RT-qPCR, immunoblotting, or fluorescent protein-fusion assays are used, then splice events can be detected, but the methods lack high-throughput capability and cannot provide real-time tracking
Solution Approach 1:
The patent replaces traditional mechanical/biochemical detection methods (RT-qPCR, immunoblotting) with a fluorescent reporter system that provides real-time optical detection of splicing events, enabling both high throughput and temporal resolution
Solution Approach 2:
The invention introduces an intein-flanked fluorescent reporter protein as an intermediary that reports splicing events through fluorescence, allowing indirect but real-time monitoring of splice events without disrupting the native splicing machinery
2Measurement precision
If conventional detection methods are used, then splice events can be identified, but they cannot provide spatiotemporal resolution for tracking disease progression
Solution Approach 1:
The patent uses fluorescent reporter proteins that emit light at specific wavelengths to indicate splicing events, providing spatial and temporal information through optical signals that can be imaged and tracked
Solution Approach 2:
The invention divides the detection function into separate modular components: the intein-flanked reporter construct inserted into specific exons, and the fluorescent reporter protein that provides the signal, allowing targeted detection of specific splice events
3Reliability
If RNA-based methods are used to detect splicing events, then mRNA presence can be detected, but they cannot reliably indicate active protein translation
Solution Approach 1:
The intein-flanked reporter protein is translated along with the protein of interest, and the intein automatically excises the reporter upon splicing, providing self-contained evidence of successful translation without requiring separate assays
Solution Approach 2:
The invention merges the detection reporter with the protein of interest through the intein-flanked construct, ensuring that detection only occurs when the mRNA is actively translated into protein rather than remaining in an arrested state
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 non-invasive, real-time tracking of specific splice events with high sensitivity and specificity, allowing for early detection of diseases like tauopathies and other neurodegenerative disorders, and can be used for therapeutic intervention.
Implementation Method 1
the expression product of the split intein - heterologous polynucleotide construct excises itself from the expression product of the specific splice product
Data Source
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AI summary
The invention provides a method for detecting a specific splice event of a gene of interest, wherein the specific splice event creates a specific splice product, which comprises an exon of interest, wherein the method comprises: (i) Inserting a split intein – heterologous polynucleotide construct into the exon of interest, wherein the split intein comprises an N-terminal splicing region upstream of the heterologous polynucleotide and a C-terminal splicing region downstream of the heterologous polynucleotide; and (ii) detecting the heterologous polynucleotide and/or the expression product of the heterologous polynucleotide. The present invention also provides the use of the split intein – heterologous polynucleotide construct, the nucleic acid encoding this construct, the vector and the host cell comprising the nucleic acid as well as a kit for detecting a specific splice event of a gene of interest.