Polychromatic dCas9 Gene Loci Detection for Multiplex Repeat Screening
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Solution Overview
Problem
Current methods for detecting specific DNA sequences, such as those associated with genetic diseases, are costly, technically complex, and time-consuming, particularly when multiple mutations need to be identified simultaneously.
Innovation Solution
A CRISPR detection platform using labeled nuclease-dead Cas9 (dCas) proteins and single guide RNA (sgRNA) complexes with different colored fluorescent proteins to detect multiple gene target loci simultaneously, enabling rapid diagnosis of genetic diseases associated with repeat expansion sequences.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If multiple gene target loci are detected using conventional methods, then detection accuracy is maintained, but detection time increases and productivity decreases
Solution Approach 1:
The patent combines multiple sgRNAs with different PAM proximal ends into a single guide RNA molecule, allowing simultaneous detection of multiple gene target loci (e.g., HBB, HBD, HBE1, HBG1, HBG2, BCL11A) in a single CRISPR reaction. This merging of detection functions into one assay dramatically reduces detection time while maintaining accuracy, directly resolving the contradiction between productivity and time loss.
Solution Approach 2:
The invention creates a universal CRISPR detection platform where a single dCas9-rtTA protein can detect multiple different gene targets by using different sgRNAs. The system's multi-functionality allows one detection system to perform what previously required multiple separate assays, improving productivity without sacrificing detection accuracy.
2Productivity
If multiple gene target loci are detected simultaneously, then productivity increases, but device complexity increases
Solution Approach 1:
The guide RNA is segmented into distinct functional regions: a first sgRNA portion targeting one gene locus and a second sgRNA portion targeting another locus, separated by PAM proximal ends. This segmentation allows the single guide RNA to direct dCas9-rtTA to multiple targets independently, enabling simultaneous detection without requiring multiple separate detection systems, thus improving productivity while managing complexity.
Solution Approach 2:
The dCas9-rtTA protein acts as an intermediary that bridges multiple target detection functions. By fusing rtTA (tetracycline-responsive transcriptional activator) to dCas9, the system creates a single molecular mediator that can be recruited to multiple different genomic loci through different sgRNA sequences, simplifying the overall system architecture while enabling multiplex detection.
3Measurement precision
If conventional detection methods are used for multiple mutations, then detection precision is maintained, but manufacturing cost increases
Solution Approach 1:
The patent merges multiple detection assays into a single CRISPR-based reaction by designing a guide RNA that contains multiple sgRNA portions with different PAM proximal ends. This consolidation eliminates the need for separate reagents, controls, and processing steps for each target, significantly reducing manufacturing costs while maintaining the detection precision achieved through the modular sgRNA design.
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 allows for the simultaneous detection of at least six different gene target loci within an hour, providing a cost-effective and efficient method for diagnosing genetic diseases like amyotrophic lateral sclerosis.
Implementation Method 1
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 2
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
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.