CRISPR Arrayed-Repeat Detection for Rapid Gene-Locus Diagnosis
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Solution Overview
Problem
Current methods for detecting genetic diseases associated with repeat expansion sequences in non-coding regions, such as FISH, are cumbersome, costly, and lack specificity, requiring lengthy assay times and multiple steps.
Innovation Solution
A CRISPR detection platform using nuclease-dead Cas9 (dCas9) constructs with fluorescent proteins bound to stem loop sequences, allowing simultaneous detection of multiple gene targets within an hour using routine laboratory procedures.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If FISH method is used to detect repeat expansion sequences, then detection capability is achieved, but assay time is excessively long (24-36 hours) and procedure is cumbersome
Solution Approach 1:
The patent extracts and eliminates the time-consuming steps from the FISH procedure. Specifically, it removes the need for lengthy DNA denaturation and oligonucleotide annealing steps by using CRISPR-Cas9 system that binds to double-stranded DNA directly, achieving detection within one hour while maintaining detection capability
Solution Approach 2:
The patent replaces the mechanical/chemical process of FISH (detergent permeabilization, heat denaturation, hybridization) with a biological system (CRISPR-Cas9 complex) that naturally binds to target DNA sequences through RNA-DNA complementarity, dramatically reducing assay time and procedural complexity
2Measurement precision
If FISH method is used for genetic disease detection, then detection is performed, but cost is high and technical difficulty is excessive
Solution Approach 1:
The patent employs a universal CRISPR-Cas9 platform that can detect multiple different genetic disease targets using the same core components (Cas9 protein, guide RNA, fluorescent marker system). By changing only the guide RNA sequence, the system can be reprogrammed to detect various repeat expansion disorders, eliminating the need for separate optimized protocols for each target and significantly reducing technical difficulty and cost
Solution Approach 2:
The patent introduces guide RNA as an intermediary molecule that mediates between the Cas9 protein and the target DNA sequence. This intermediary allows for easy reprogramming of detection targets by simply changing the guide RNA sequence, making the system highly adaptable and technologically accessible without requiring complex optimization for each new target
3Reliability
If FISH method is used, then detection is performed, but specificity is insufficient and multiple steps are required
Solution Approach 1:
The patent applies local quality by designing the guide RNA with a specific 20-nucleotide sequence that is complementary to the target DNA sequence, creating highly localized and specific binding. The CRISPR-Cas9 complex recognizes and binds only to the precise target sequence through RNA-DNA base pairing, ensuring high specificity while simplifying the overall procedure to a single binding step rather than multiple FISH steps
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 platform enables rapid and specific diagnosis of genetic diseases by detecting repeat expansion sequences in non-coding regions with improved efficiency and cost-effectiveness, capable of identifying at least six different gene loci simultaneously.
Implementation Method 1
A CRISPR detection platform using nuclease-dead Cas9 (dCas9) constructs with fluorescent proteins bound to stem loop sequences
Implementation Method 2
Cas9 can be guided to specific sites in the human genome through base-pair complementation between a 20 nucleotide guide region of an engineered single guide RNA (sgRNA) and a genomic target sequence
Implementation Method 3
dCas9 constructs are also contemplated as having fluorescent proteins bound to any or all stem loop sequences, wherein detection of a plurality of dCas9 constructs having different colored fluorescent proteins can simultaneously detect at least six (6) different gene target loci
Data Source
Figure 1A~1C
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AI summary
A C9orf72 DNA repeat expansion can be detected using a CRISPR Arrayed Repeat Detection System (CARDS). Based upon the compositions and methods supporting this platform primary cell cultures and/or blood cell smears can be tested under conventional clinical diagnostic laboratory conditions to diagnose genetically-based diseases having DNA repeat expansions, including but not limited to ALS. dCas9 constructs are also contemplated as having fluorescent proteins bound to any or all stem loop sequences, wherein detection of a plurality of dCas9 constructs having different colored fluorescent proteins can simultaneously detect at least six (6) different gene target loci.